Bio


Wah Chiu received his BA in Physics (1969) and PhD in Biophysics (1975) from the University of California, Berkeley. He is a professor in the Department of Bioengineering, Department of Microbiology and Immunology and the SLAC National Accelerator Laboratory at Stanford University. He is a pioneer in methodology development for electron cryo-microscopy. His work has made multiple transformational contributions in developing single particle electron cryo-microscopy as a tool for the structural determination of molecular machines at atomic resolution.

For three decades, Dr. Chiu directs a NIH funded 3DEM Resource Center. He has solved many cryo-EM structures including viruses, chaperonins, membrane proteins, ion channels, cytoskeleton protein complexes, protein-DNA complexes, DNA and RNA in collaboration with many scientists around the world. His 3DEM Resource Center continues to establish high standard testing and characterization protocols for cryoEM instrumentation and to develop new image processing and modeling algorithms for cryo-EM structure determination.

Dr. Chiu’s research, collaboration and training efforts have been recognized by his elected membership to the Academia Sinica, Taiwan (2008), and the United States National Academy of Sciences (2012), in addition to several honors including the Distinguished Science Award from the Microscopy Society of America (2014) and the Honorary Doctorate of Philosophy from the University of Helsinki, Finland (2014).

Academic Appointments


Honors & Awards


  • Elected Member, United States National Academy of Sciences (2012)
  • Elected Academician, Academia Sinica, Taiwan (2008)
  • Distinguished Scientist Award for the Biological Sciences, Microscopy Society of America (2014)
  • Honorary Doctorate of Philosophy, University of Helsinki, Finland (2014)
  • Barbara and Corbin J. Robertson Jr. Presidential Award for Excellence in Education, Baylor College of Medicine (2015)
  • Elected Member, The Academy of Medicine, Engineering, and Science of Texas (2013)
  • Distinguished Faculty Award, Baylor College of Medicine Alumni Association (2013)
  • Distinguished Service Professorship, Baylor College of Medicine (2010)
  • Achievement Award, Society of Chinese Bioscientists in America Houston Chapter (2011)
  • Presidential Award, American Academy of Nanomedicine (2006)
  • Research Fellow, Japan Society for the Promotion of Science (1999)
  • Alexander von Humboldt Research Prize, Alexander von Humboldt Foundation (1996)
  • Guggenheim Fellow, Guggenheim Foundation (1986)
  • Presidential Scholar, Electron Microscopy Society of America (1974)
  • Award of Merit, Oakland City Council (1972)

Boards, Advisory Committees, Professional Organizations


  • Member, Scientific Advisory Board, Biozentrum, Universität Basel, Basel, Switzerland (2016 - Present)
  • Member, Scientific Advisory Board, BioXFEL Center, University of Buffalo (2014 - Present)
  • Member, Scientific Advisory Board, Institute of Biological Chemistry Institute, Academia Sinica, Taiwan (2011 - Present)
  • Member, Advisory Committee, World-wide Protein Data Bank (wwPDB) (2010 - Present)
  • Member, Scientific Advisory Board, RCSB Protein Data Bank (2005 - Present)
  • Member, Panel for SystemsX, Research Council of the Swiss National Science Foundation (2007 - 2016)
  • Member, Scientific Advisory Board, Division of Structural Biology, St. Jude Children’s Research Hospital (2015 - 2016)
  • Member, Scientific Advisory Board, Max Planck Institute of Biochemistry in Martinsried, Germany (2013 - 2017)
  • Chair, Expert Panel for Biomedical Engineering & Life Sciences Cluster, Singapore Ministry of Education (2012 - 2015)
  • Member, Scientific Advisory Board, Michael E. DeBakey VA Medical Center, Houston (2012 - 2017)

Professional Education


  • Ph.D., University of California, Berkeley, Biophysics (1975)
  • B.A., University of California, Berkeley, Physics (1969)

Current Research and Scholarly Interests


My research includes methodology improvements in single particle cryo-EM for atomic resolution structure determination of molecules and molecular machines, as well as in cryo-ET of cells and organelles towards subnanometer resolutions. We collaborate with many researchers around the country and outside the USA on understanding biological processes such as protein folding, virus assembly and disassembly, pathogen-host interactions, signal transduction, and transport across cytosol and membranes.

Projects


  • Cryo-EM of Molecular Machines, Stanford University (1/1/2018 - Present)

    Use cryo-EM to determine atomic structures of channels, pumps, transporters, chaperonins, protein degradation machines, and viruses in different functional states in conjunction with biochemical and physiological characterizations.

    Location

    Stanford, CA

  • Unified Data Resource for 3DEM, Stanford University (1/1/2018)

    Unified Data Resource for 3DEM

    Location

    Stanford, CA

    Collaborators

    • Helen Berman, Professor, Rutgers University
  • From structure to therapy for Huntington’s disease, Stanford University (1/1/2018)

    Location

    Stanford, CA

  • Pathogen-Host interactions studied by cryo-EM and cryo-ET, Stanford University (1/1/2018 - Present)

    Our interests span across DNA and RNA viruses infecting from bacteria, archaea and mammals.

    Location

    Stanford, CA

  • DNA and RAN Origami Characterization, Stanford University (1/1/2018)

    Location

    Stanford, CA

  • Atomic resolution single particle cryoEM, Stanford University

    Location

    Stanford, CA

2019-20 Courses


Stanford Advisees


All Publications


  • Measurement of atom resolvability in cryo-EM maps with Q-scores. Nature methods Pintilie, G., Zhang, K., Su, Z., Li, S., Schmid, M. F., Chiu, W. 2020

    Abstract

    Cryogenic electron microscopy (cryo-EM) maps are now at the point where resolvability of individual atoms can be achieved. However, resolvability is not necessarily uniform throughout the map. We introduce a quantitative parameter to characterize the resolvability of individual atoms in cryo-EM maps, the map Q-score. Q-scores can be calculated for atoms in proteins, nucleic acids, water, ligands and other solvent atoms, using models fitted to or derived from cryo-EM maps. Q-scores can also be averaged to represent larger features such as entire residues and nucleotides. Averaged over entire models, Q-scores correlate very well with the estimated resolution of cryo-EM maps for both protein and RNA. Assuming the models they are calculated from are well fitted to the map, Q-scores can be used as a measure of resolvability in cryo-EM maps at various scales, from entire macromolecules down to individual atoms. Q-score analysis of multiple cryo-EM maps of the same proteins derived from different laboratories confirms the reproducibility of structural features from side chains down to water and ion atoms.

    View details for DOI 10.1038/s41592-020-0731-1

    View details for PubMedID 32042190

  • Cryo-EM structure of a 40 kDa SAM-IV riboswitch RNA at 3.7 Å resolution Nat Commun Li, K., L, S., Kappel, K., Pintilie, G., Su, Z., Mou, T., Schmid, M. F., Das, R., Chiu, W. 2019; 10: 5511
  • Visualizing Individual RuBisCO and Its Assembly into Carboxysomes in Marine Cyanobacteria by Cryo-Electron Tomography JOURNAL OF MOLECULAR BIOLOGY Dai, W., Chen, M., Myers, C., Ludtke, S. J., Pettitt, B., King, J. A., Schmid, M. F., Chiu, W. 2018; 430 (21): 4156–67

    Abstract

    Cyanobacteria are photosynthetic organisms responsible for ~25% of the organic carbon fixation on earth. A key step in carbon fixation is catalyzed by ribulose bisphosphate carboxylase/oxygenase (RuBisCO), the most abundant enzyme in the biosphere. Applying Zernike phase-contrast electron cryo-tomography and automated annotation, we identified individual RuBisCO molecules and their assembly intermediates leading to the formation of carboxysomes inside Syn5 cyanophage infected cyanobacteria Synechococcus sp. WH8109 cells. Surprisingly, more RuBisCO molecules were found to be present as cytosolic free-standing complexes or clusters than as packaged assemblies inside carboxysomes. Cytosolic RuBisCO clusters and partially assembled carboxysomes identified in the cell tomograms support a concurrent assembly model involving both the protein shell and the enclosed RuBisCO. In mature carboxysomes, RuBisCO is neither randomly nor strictly icosahedrally packed within protein shells of variable sizes. A time-averaged molecular dynamics simulation showed a semi-liquid probability distribution of the RuBisCO in carboxysomes and correlated well with carboxysome subtomogram averages. Our structural observations reveal the various stages of RuBisCO assemblies, which could be important for understanding cellular function.

    View details for DOI 10.1016/j.jmb.2018.08.013

    View details for Web of Science ID 000448493400020

    View details for PubMedID 30138616

    View details for PubMedCentralID PMC6252266

  • The 3.5-A CryoEM Structure of Nanodisc-Reconstituted Yeast Vacuolar ATPase Vo Proton Channel. Mol Cell Roh, S. H., Stam, N. J., Hryc, C. F., Couoh-Cardel, S., Pintilie, G., Chiu, W., Wilkens, S. 2018; 69 (6): 993-1004
  • Accurate model annotation of a near-atomic resolution cryo-EM map PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hryc, C. F., Chen, D., Afonine, P. V., Jakana, J., Wang, Z., Haase-Pettingell, C., Jiang, W., Adams, P. D., King, J. A., Schmid, M. F., Chiu, W. 2017; 114 (12): 3103-3108

    Abstract

    Electron cryomicroscopy (cryo-EM) has been used to determine the atomic coordinates (models) from density maps of biological assemblies. These models can be assessed by their overall fit to the experimental data and stereochemical information. However, these models do not annotate the actual density values of the atoms nor their positional uncertainty. Here, we introduce a computational procedure to derive an atomic model from a cryo-EM map with annotated metadata. The accuracy of such a model is validated by a faithful replication of the experimental cryo-EM map computed using the coordinates and associated metadata. The functional interpretation of any structural features in the model and its utilization for future studies can be made in the context of its measure of uncertainty. We applied this protocol to the 3.3-Å map of the mature P22 bacteriophage capsid, a large and complex macromolecular assembly. With this protocol, we identify and annotate previously undescribed molecular interactions between capsid subunits that are crucial to maintain stability in the absence of cementing proteins or cross-linking, as occur in other bacteriophages.

    View details for DOI 10.1073/pnas.1621152114

    View details for Web of Science ID 000396893600057

    View details for PubMedID 28270620

  • Gating machinery of InsP3R channels revealed by electron cryomicroscopy Gating machinery of InsP3R channels revealed by electron cryomicroscopy Fan, G., Baker, M. L., Wang, Z., Baker, M. R., Sinyagovskiy, P. A., Chiu, W., Ludtke, S. J., Serysheva, I. I. 2015; 527 (7578): 336-341

    View details for DOI 10.1038/nature15249

  • Visualizing virus assembly intermediates inside marine cyanobacteria. Nature Dai, W., Fu, C., Raytcheva, D., Flanagan, J., Khant, H. A., Liu, X., Rochat, R. H., Haase-Pettingell, C., Piret, J., Ludtke, S. J., Nagayama, K., Schmid, M. F., King, J. A., Chiu, W. 2013; 502 (7473): 707–10

    Abstract

    Cyanobacteria are photosynthetic organisms responsible for ∼25% of organic carbon fixation on the Earth. These bacteria began to convert solar energy and carbon dioxide into bioenergy and oxygen more than two billion years ago. Cyanophages, which infect these bacteria, have an important role in regulating the marine ecosystem by controlling cyanobacteria community organization and mediating lateral gene transfer. Here we visualize the maturation process of cyanophage Syn5 inside its host cell, Synechococcus, using Zernike phase contrast electron cryo-tomography (cryoET). This imaging modality yields dramatic enhancement of image contrast over conventional cryoET and thus facilitates the direct identification of subcellular components, including thylakoid membranes, carboxysomes and polyribosomes, as well as phages, inside the congested cytosol of the infected cell. By correlating the structural features and relative abundance of viral progeny within cells at different stages of infection, we identify distinct Syn5 assembly intermediates. Our results indicate that the procapsid releases scaffolding proteins and expands its volume at an early stage of genome packaging. Later in the assembly process, we detected full particles with a tail either with or without an additional horn. The morphogenetic pathway we describe here is highly conserved and was probably established long before that of double-stranded DNA viruses infecting more complex organisms.

    View details for DOI 10.1038/nature12604

    View details for PubMedID 24107993

    View details for PubMedCentralID PMC3984937

  • Mechanism of folding chamber closure in a group II chaperonin NATURE Zhang, J., Baker, M. L., Schroeder, G. F., Douglas, N. R., Reissmann, S., Jakana, J., Dougherty, M., Fu, C. J., Levitt, M., Ludtke, S. J., Frydman, J., Chiu, W. 2010; 463 (7279): 379-U130

    Abstract

    Group II chaperonins are essential mediators of cellular protein folding in eukaryotes and archaea. These oligomeric protein machines, approximately 1 megadalton, consist of two back-to-back rings encompassing a central cavity that accommodates polypeptide substrates. Chaperonin-mediated protein folding is critically dependent on the closure of a built-in lid, which is triggered by ATP hydrolysis. The structural rearrangements and molecular events leading to lid closure are still unknown. Here we report four single particle cryo-electron microscopy (cryo-EM) structures of Mm-cpn, an archaeal group II chaperonin, in the nucleotide-free (open) and nucleotide-induced (closed) states. The 4.3 A resolution of the closed conformation allowed building of the first ever atomic model directly from the single particle cryo-EM density map, in which we were able to visualize the nucleotide and more than 70% of the side chains. The model of the open conformation was obtained by using the deformable elastic network modelling with the 8 A resolution open-state cryo-EM density restraints. Together, the open and closed structures show how local conformational changes triggered by ATP hydrolysis lead to an alteration of intersubunit contacts within and across the rings, ultimately causing a rocking motion that closes the ring. Our analyses show that there is an intricate and unforeseen set of interactions controlling allosteric communication and inter-ring signalling, driving the conformational cycle of group II chaperonins. Beyond this, we anticipate that our methodology of combining single particle cryo-EM and computational modelling will become a powerful tool in the determination of atomic details involved in the dynamic processes of macromolecular machines in solution.

    View details for DOI 10.1038/nature08701

    View details for Web of Science ID 000273748100049

    View details for PubMedID 20090755

    View details for PubMedCentralID PMC2834796

  • Inhibition mechanisms of AcrF9, AcrF8, and AcrF6 against type I-F CRISPR-Cas complex revealed by cryo-EM. Proceedings of the National Academy of Sciences of the United States of America Zhang, K., Wang, S., Li, S., Zhu, Y., Pintilie, G. D., Mou, T., Schmid, M. F., Huang, Z., Chiu, W. 2020

    Abstract

    Prokaryotes and viruses have fought a long battle against each other. Prokaryotes use CRISPR-Cas-mediated adaptive immunity, while conversely, viruses evolve multiple anti-CRISPR (Acr) proteins to defeat these CRISPR-Cas systems. The type I-F CRISPR-Cas system in Pseudomonas aeruginosa requires the crRNA-guided surveillance complex (Csy complex) to recognize the invading DNA. Although some Acr proteins against the Csy complex have been reported, other relevant Acr proteins still need studies to understand their mechanisms. Here, we obtain three structures of previously unresolved Acr proteins (AcrF9, AcrF8, and AcrF6) bound to the Csy complex using electron cryo-microscopy (cryo-EM), with resolution at 2.57 A, 3.42 A, and 3.15 A, respectively. The 2.57-A structure reveals fine details for each molecular component within the Csy complex as well as the direct and water-mediated interactions between proteins and CRISPR RNA (crRNA). Our structures also show unambiguously how these Acr proteins bind differently to the Csy complex. AcrF9 binds to key DNA-binding sites on the Csy spiral backbone. AcrF6 binds at the junction between Cas7.6f and Cas8f, which is critical for DNA duplex splitting. AcrF8 binds to a distinct position on the Csy spiral backbone and forms interactions with crRNA, which has not been seen in other Acr proteins against the Csy complex. Our structure-guided mutagenesis and biochemistry experiments further support the anti-CRISPR mechanisms of these Acr proteins. Our findings support the convergent consequence of inhibiting degradation of invading DNA by these Acr proteins, albeit with different modes of interactions with the type I-F CRISPR-Cas system.

    View details for DOI 10.1073/pnas.1922638117

    View details for PubMedID 32170016

  • Structure of the G protein chaperone and guanine nucleotide exchange factor Ric-8A bound to Galphai1. Nature communications McClelland, L. J., Zhang, K., Mou, T., Johnston, J., Yates-Hansen, C., Li, S., Thomas, C. J., Doukov, T. I., Triest, S., Wohlkonig, A., Tall, G. G., Steyaert, J., Chiu, W., Sprang, S. R. 2020; 11 (1): 1077

    Abstract

    Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates heterotrimeric G protein alpha subunits (Galpha) and serves as an essential Galpha chaperone. Mechanisms by which Ric-8A catalyzes these activities, which are stimulated by Casein Kinase II phosphorylation, are unknown. We report the structure of the nanobody-stabilized complex of nucleotide-free Galpha bound to phosphorylated Ric-8A at near atomic resolution by cryo-electron microscopy and X-ray crystallography. The mechanism of Ric-8A GEF activity differs considerably from that employed by G protein-coupled receptors at the plasma membrane. Ric-8A engages a specific conformation of Galpha at multiple interfaces to form a complex that is stabilized by phosphorylation within a Ric-8A segment that connects two Galpha binding sites. The C-terminus of Galpha is ejected from its beta sheet core, thereby dismantling the GDP binding site. Ric-8A binds to the exposed Galpha beta sheet and switch II to stabilize the nucleotide-free state of Galpha.

    View details for DOI 10.1038/s41467-020-14943-4

    View details for PubMedID 32103024

  • Ultra-thermostable RNA nanoparticles for solubilizing and high-yield loading of paclitaxel for breast cancer therapy. Nature communications Guo, S., Vieweger, M., Zhang, K., Yin, H., Wang, H., Li, X., Li, S., Hu, S., Sparreboom, A., Evers, B. M., Dong, Y., Chiu, W., Guo, P. 2020; 11 (1): 972

    Abstract

    Paclitaxel is widely used in cancer treatments, but poor water-solubility and toxicity raise serious concerns. Here we report an RNA four-way junction nanoparticle with ultra-thermodynamic stability to solubilize and load paclitaxel for targeted cancer therapy. Each RNA nanoparticle covalently loads twenty-four paclitaxel molecules as aprodrug. The RNA-paclitaxel complex is structurally rigid and stable, demonstrated by the sub-nanometer resolution imaging of cryo-EM. Using RNA nanoparticles as carriers increases the water-solubility of paclitaxel by 32,000-fold. Intravenous injections of RNA-paclitaxel nanoparticles with specific cancer-targeting ligand dramatically inhibit breast cancer growth, with nearly undetectable toxicity and immune responses in mice. No fatalities are observed at a paclitaxel dose equal to the reported LD50. The use of ultra-thermostable RNA nanoparticles to deliver chemical prodrugs addresses issues with RNA unfolding and nanoparticle dissociation after high-density drug loading. This finding provides a stable nano-platform for chemo-drug delivery as well as an efficient method to solubilize hydrophobic drugs.

    View details for DOI 10.1038/s41467-020-14780-5

    View details for PubMedID 32080195

  • Cryogenic Superresolution Fluorescence Correlated with Cryogenic Electron Tomography: Combining Specific Labeling and High Resolution Dahlberg, P. D., Saurabh, S., Wang, J., Sartor, A. M., Chiu, W., Shapiro, L., Moerner, W. E. CELL PRESS. 2020: 20A–21A
  • TrkA undergoes a tetramer-to-dimer conversion to open TrkH which enables changes in membrane potential. Nature communications Zhang, H., Pan, Y., Hu, L., Hudson, M. A., Hofstetter, K. S., Xu, Z., Rong, M., Wang, Z., Prasad, B. V., Lockless, S. W., Chiu, W., Zhou, M. 2020; 11 (1): 547

    Abstract

    TrkH is a bacterial ion channel implicated in K+ uptake and pH regulation. TrkH assembles with its regulatory protein, TrkA, which closes the channel when bound to ADP and opens it when bound to ATP. However, it is unknown how nucleotides control the gating of TrkH through TrkA. Here we report the structures of the TrkH-TrkA complex in the presence of ADP or ATP. TrkA forms a tetrameric ring when bound to ADP and constrains TrkH to a closed conformation. The TrkA ring splits into two TrkA dimers in the presence of ATP and releases the constraints on TrkH, resulting in an open channel conformation. Functional studies show that both the tetramer-to-dimer conversion of TrkA and the loss of constraints on TrkH are required for channel gating. In addition, deletion of TrkA in Escherichia coli depolarizes the cell, suggesting that the TrkH-TrkA complex couples changes in intracellular nucleotides to membrane potential.

    View details for DOI 10.1038/s41467-019-14240-9

    View details for PubMedID 31992706

  • Evolving data standards for cryo-EM structures. Structural dynamics (Melville, N.Y.) Lawson, C. L., Berman, H. M., Chiu, W. 2020; 7 (1): 014701

    Abstract

    Electron cryo-microscopy (cryo-EM) is increasingly being used to determine 3D structures of a broad spectrum of biological specimens from molecules to cells. Anticipating this progress in the early 2000s, an international collaboration of scientists with expertise in both cryo-EM and structure data archiving was established (EMDataResource, previously known as EMDataBank). The major goals of the collaboration have been twofold: to develop the necessary infrastructure for archiving cryo-EM-derived density maps and models, and to promote development of cryo-EM structure validation standards. We describe how cryo-EM data archiving and validation have been developed and jointly coordinated for the Electron Microscopy Data Bank and Protein Data Bank archives over the past two decades, as well as the impact of evolving technology on data standards. Just as for X-ray crystallography and nuclear magnetic resonance, engaging the scientific community via workshops and challenging activities has played a central role in developing recommendations and requirements for the cryo-EM structure data archives.

    View details for DOI 10.1063/1.5138589

    View details for PubMedID 32002441

    View details for PubMedCentralID PMC6980868

  • Arrangement of the Polymerase Complexes inside a Nine-Segmented dsRNA Virus. Structure (London, England : 1993) Kaelber, J. T., Jiang, W., Weaver, S. C., Auguste, A. J., Chiu, W. 2020

    Abstract

    Members of the family Reoviridae package several copies of the viral polymerase complex into their capsid to carry out replication and transcription within viral particles. Classical single-particle reconstruction encounters difficulties resolving structures such as the intraparticle polymerase complex because refinement can converge to an incorrect map and because the map could depict a nonrepresentative subset of particles or an average of heterogeneous particles. Using the nine-segmented Fako virus, we tested hypotheses for the arrangement and number of polymerase complexes within the virion by measuring how well each hypothesis describes the set of cryoelectron microscopy images of individual viral particles. We find that the polymerase complex in Fako virus binds at ten possible sites despite having only nine genome segments. A single asymmetric configuration describes the arrangement of these complexes in both virions and genome-free capsids. Similarities between the arrangements of Reoviridae with 9, 10, and 11 segments indicate the generalizability of this architecture.

    View details for DOI 10.1016/j.str.2020.01.011

    View details for PubMedID 32049031

  • Federating Structural Models and Data: Outcomes from A Workshop on Archiving Integrative Structures STRUCTURE Berman, H. M., Adams, P. D., Bonvin, A. A., Burley, S. K., Carragher, B., Chiu, W., DiMaio, F., Ferrin, T. E., Gabanyi, M. J., Goddard, T. D., Griffin, P. R., Haas, J., Hanke, C. A., Hoch, J. C., Hummer, G., Kurisu, G., Lawson, C. L., Leitner, A., Markley, J. L., Meiler, J., Montelione, G. T., Phillips, G. N., Prisner, T., Rappsilber, J., Schriemer, D. C., Schwede, T., Seidel, C. M., Strutzenberg, T. S., Svergun, D. I., Tajkhorshid, E., Trewhella, J., Vallat, B., Velankar, S., Vuister, G. W., Webb, B., Westbrook, J. D., White, K. L., Sali, A. 2019; 27 (12): 1745–59

    Abstract

    Structures of biomolecular systems are increasingly computed by integrative modeling. In this approach, a structural model is constructed by combining information from multiple sources, including varied experimental methods and prior models. In 2019, a Workshop was held as a Biophysical Society Satellite Meeting to assess progress and discuss further requirements for archiving integrative structures. The primary goal of the Workshop was to build consensus for addressing the challenges involved in creating common data standards, building methods for federated data exchange, and developing mechanisms for validating integrative structures. The summary of the Workshop and the recommendations that emerged are presented here.

    View details for DOI 10.1016/j.str.2019.11.002

    View details for Web of Science ID 000500745700005

    View details for PubMedID 31780431

  • Unravelling Degradation Mechanisms and Atomic Structure of Organic-Inorganic Halide Perovskites by Cryo-EM JOULE Li, Y., Zhou, W., Li, Y., Huang, W., Zhang, Z., Chen, G., Wang, H., Wu, G., Rolston, N., Vila, R., Chiu, W., Cui, Y. 2019; 3 (11): 2854–66
  • Structural basis of amino acid surveillance by higher-order tRNA-mRNA interactions. Nature structural & molecular biology Li, S., Su, Z., Lehmann, J., Stamatopoulou, V., Giarimoglou, N., Henderson, F. E., Fan, L., Pintilie, G. D., Zhang, K., Chen, M., Ludtke, S. J., Wang, Y., Stathopoulos, C., Chiu, W., Zhang, J. 2019

    Abstract

    Amino acid availability in Gram-positive bacteria is monitored by T-box riboswitches. T-boxes directly bind tRNAs, assess their aminoacylation state, and regulate the transcription or translation of downstream genes to maintain nutritional homeostasis. Here, we report cocrystal and cryo-EM structures of Geobacillus kaustophilus and Bacillus subtilis T-box-tRNA complexes, detailing their multivalent, exquisitely selective interactions. The T-box forms a U-shaped molecular vise that clamps the tRNA, captures its 3' end using an elaborate 'discriminator' structure, and interrogates its aminoacylation state using a steric filter fashioned from a wobble base pair. In the absence of aminoacylation, T-boxes clutch tRNAs and form a continuously stacked central spine, permitting transcriptional readthrough or translation initiation. A modeled aminoacyl disrupts tRNA-T-box stacking, severing the central spine and blocking gene expression. Our data establish a universal mechanism of amino acid sensing on tRNAs and gene regulation by T-box riboswitches and exemplify how higher-order RNA-RNA interactions achieve multivalency and specificity.

    View details for DOI 10.1038/s41594-019-0326-7

    View details for PubMedID 31740854

  • Cryo-electron microscopy targets in CASP13: Overview and evaluation of results PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS Kryshtafovych, A., Malhotra, S., Monastyrskyy, B., Cragnolini, T., Joseph, A., Chiu, W., Topf, M. 2019; 87 (12): 1128–40

    Abstract

    Structures of seven CASP13 targets were determined using cryo-electron microscopy (cryo-EM) technique with resolution between 3.0 and 4.0 Å. We provide an overview of the experimentally derived structures and describe results of the numerical evaluation of the submitted models. The evaluation is carried out by comparing coordinates of models to those of reference structures (CASP-style evaluation), as well as checking goodness-of-fit of modeled structures to the cryo-EM density maps. The performance of contributing research groups in the CASP-style evaluation is measured in terms of backbone accuracy, all-atom local geometry and similarity of inter-subunit interfaces. The results on the cryo-EM targets are compared with those on the whole set of eighty CASP13 targets. A posteriori refinement of the best models in their corresponding cryo-EM density maps resulted in structures that are very close to the reference structure, including some regions with better fit to the density.

    View details for DOI 10.1002/prot.25817

    View details for Web of Science ID 000491655700001

    View details for PubMedID 31576602

  • Segmentation and Comparative Modeling in an 8.6-angstrom Cryo-EM Map of the Singapore Grouper Iridovirus STRUCTURE Pintilie, G., Chen, D., Bich Ngoc Tran, Jakana, J., Wu, J., Hew, C., Chiu, W. 2019; 27 (10): 1561-+

    Abstract

    SGIV, or Singapore grouper iridovirus, is a large double-stranded DNA virus, reaching a diameter of 220 nm and packaging a genome of 140 kb. We present a 3D cryoelectron microscopy (cryo-EM) icosahedral reconstruction of SGIV determined at 8.6-Å resolution. It reveals several layers including a T = 247 icosahedral outer coat, anchor proteins, a lipid bilayer, and the encapsidated DNA. A new segmentation tool, iSeg, was applied to extract these layers from the reconstructed map. The outer coat was further segmented into major and minor capsid proteins. None of the proteins extracted by segmentation have known atomic structures. We generated models for the major coat protein using three comparative modeling tools, and evaluated each model using the cryo-EM map. Our analysis reveals a new architecture in the Iridoviridae family of viruses. It shares similarities with others in the same family, e.g., Chilo iridescent virus, but also shows new features of the major and minor capsid proteins.

    View details for DOI 10.1016/j.str.2019.08.002

    View details for Web of Science ID 000488523700009

    View details for PubMedID 31447288

    View details for PubMedCentralID PMC6853598

  • Redox Engineering of Cytochrome c using DNA Nanostructure-Based Charged Encapsulation and Spatial Control. ACS applied materials & interfaces Ge, Z., Su, Z., Simmons, C. R., Li, J., Jiang, S., Li, W., Yang, Y., Liu, Y., Chiu, W., Fan, C., Yan, H. 2019; 11 (15): 13874–80

    Abstract

    Three-dimensional (3D) DNA nanostructures facilitate the directed self-assembly of various objects with designed patterns with nanometer scale addressability. Here, we report the enhancement of cytochrome c (cyt c) redox activity by using a designed 3D DNA nanostructure attached to a gold electrode to spatially control the position of cyt c within the tetrahedral framework. Charged encapsulation and spatial control result in the significantly increased redox potential and enhanced electron transfer of this redox protein when compared to cyt c directly adsorbed on the gold surface. Two different protein attachment sites on one double stranded edge of a DNA tetrahedron were used to position cyt c inside and outside of the cage. Cyt c at both binding sites show similar redox potential shift and only slight difference in the electron transfer rate, both orders of magnitude faster than the cases when the protein was directly deposited on the gold electrode, likely due to an effective electron transfer pathway provided by the stabilization effect of the protein created by the DNA framework. This study shows great potential of using structural DNA nanotechnology for spatial control of protein positioning on electrode, which opens new routes to engineer redox proteins and interface microelectronic devices with biological function.

    View details for PubMedID 29939710

  • Coupling of ssRNA cleavage with DNase activity in type III-A CRISPR-Csm revealed by cryo-EM and biochemistry CELL RESEARCH Guo, M., Zhang, K., Zhu, Y., Pintilie, G. D., Guan, X., Li, S., Schmid, M. F., Ma, Z., Chiu, W., Huang, Z. 2019; 29 (4): 305–12
  • The Chaperonin TRiC/CCT Associates with Prefoldin through a Conserved Electrostatic Interface Essential for Cellular Proteostasis. Cell Gestaut, D., Roh, S. H., Ma, B., Pintilie, G., Joachimiak, L. A., Leitner, A., Walzthoeni, T., Aebersold, R., Chiu, W., Frydman, J. 2019

    Abstract

    Maintaining proteostasis in eukaryotic protein folding involves cooperation of distinct chaperone systems. To understand how the essential ring-shaped chaperonin TRiC/CCT cooperates with the chaperone prefoldin/GIMc (PFD), we integrate cryoelectron microscopy (cryo-EM), crosslinking-mass-spectrometry and biochemical and cellular approaches to elucidate the structural and functional interplay between TRiC/CCT and PFD. We find these hetero-oligomeric chaperones associate in a defined architecture, through a conserved interface of electrostatic contacts that serves as a pivot point for a TRiC-PFD conformational cycle. PFD alternates between an open "latched" conformation and a closed "engaged" conformation that aligns the PFD-TRiC substrate binding chambers. PFD can act after TRiC bound its substrates to enhance the rate and yield of the folding reaction, suppressing non-productive reaction cycles. Disrupting the TRiC-PFD interaction invivo is strongly deleterious, leading to accumulation of amyloid aggregates. The supra-chaperone assembly formed by PFD and TRiC is essential to prevent toxic conformations and ensure effective cellular proteostasis.

    View details for PubMedID 30955883

  • Cryo-EM structures of Helicobacter pylori vacuolating cytotoxin A oligomeric assemblies at near-atomic resolution. Proceedings of the National Academy of Sciences of the United States of America Zhang, K., Zhang, H., Li, S., Pintilie, G. D., Mou, T., Gao, Y., Zhang, Q., van den Bedem, H., Schmid, M. F., Au, S. W., Chiu, W. 2019

    Abstract

    Human gastric pathogen Helicobacter pylori (H. pylori) is the primary risk factor for gastric cancer and is one of the most prevalent carcinogenic infectious agents. Vacuolating cytotoxin A (VacA) is a key virulence factor secreted by H. pylori and induces multiple cellular responses. Although structural and functional studies of VacA have been extensively performed, the high-resolution structure of a full-length VacA protomer and the molecular basis of its oligomerization are still unknown. Here, we use cryoelectron microscopy to resolve 10 structures of VacA assemblies, including monolayer (hexamer and heptamer) and bilayer (dodecamer, tridecamer, and tetradecamer) oligomers. The models of the 88-kDa full-length VacA protomer derived from the near-atomic resolution maps are highly conserved among different oligomers and show a continuous right-handed beta-helix made up of two domains with extensive domain-domain interactions. The specific interactions between adjacent protomers in the same layer stabilizing the oligomers are well resolved. For double-layer oligomers, we found short- and/or long-range hydrophobic interactions between protomers across the two layers. Our structures and other previous observations lead to a mechanistic model wherein VacA hexamer would correspond to the prepore-forming state, and the N-terminal region of VacA responsible for the membrane insertion would undergo a large conformational change to bring the hydrophobic transmembrane region to the center of the oligomer for the membrane channel formation.

    View details for PubMedID 30894496

  • Structure of Calcarisporiella thermophila Hsp104 Disaggregase that Antagonizes Diverse Proteotoxic Misfolding Events STRUCTURE Michalska, K., Zhang, K., March, Z. M., Hatzos-Skintges, C., Pintilie, G., Bigelow, L., Castellano, L. M., Miles, L. J., Jackrel, M. E., Chuang, E., Jedrzejczak, R., Shorter, J., Chiu, W., Joachimiak, A. 2019; 27 (3): 449-+
  • Coupling of ssRNA cleavage with DNase activity in type III-A CRISPR-Csm revealed by cryo-EM and biochemistry. Cell research Guo, M., Zhang, K., Zhu, Y., Pintilie, G. D., Guan, X., Li, S., Schmid, M. F., Ma, Z., Chiu, W., Huang, Z. 2019

    Abstract

    The type III CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated genes) systems are bacterially encoded adaptive immune systems for defense against invading nucleic acids. They accomplish this task through the coordinated cleavage of invading substrates of single-stranded RNA and DNA (ssDNA and ssRNA) by the Csm (type III-A) or Cmr (type III-B) effector complexes. The ssRNA is complementarily bound to the CRISPR RNA (crRNA). However, the structural basis for the DNase and RNase activation of the Csm nucleoprotein complex is largely unknown. Here we report cryo-EM structures of the Csm-crRNA complex, with or without target ssRNA, at near-atomic resolution. Our cryo-EM maps allow us to build atomic models of the key macromolecular components, including Cas10, Csm2, Csm3, Csm4, crRNA and the invading ssRNA. Our structure resolves unambiguously the stoichiometry and tertiary structures of the Csm protein complex and the interactions between protein components and the crRNA/ssRNA. Interestingly, the new atomic structures of the Csm proteins presented here are similar to those of previously known Csm proteins in other species despite their low sequence similarity. Our combined structural and biochemical data suggest that ssRNA cleavage is preferentially carried out near its 5'-end, that the extent of interactions among the ssRNA, crRNA and the protein components regulates the DNase activity of the Csm complex, and that the 3' flanking sequence of ssRNA activates the Cas10 DNase activity allosterically.

    View details for PubMedID 30814678

  • Outcomes of the Cryo-EM Map and Model Challenges Lawson, C. L., Kryshtafovych, A., Pintilie, G., Berman, H. M., Chiu, W. CELL PRESS. 2019: 160A
  • Automated Sequence Design of 3D Polyhedral Wireframe DNA Origami with Honeycomb Edges ACS NANO Jun, H., Shepherd, T. R., Zhang, K., Bricker, W. P., Li, S., Chiu, W., Bathe, M. 2019; 13 (2): 2083–93

    Abstract

    3D polyhedral wireframe DNA nanoparticles (DNA-NPs) fabricated using scaffolded DNA origami offer complete and independent control over NP size, structure, and asymmetric functionalization on the 10-100 nm scale. However, the complex DNA sequence design needed for the synthesis of these versatile DNA-NPs has limited their widespread use to date. While the automated sequence design algorithms DAEDALUS and vHelix-BSCOR apply to DNA-NPs synthesized using either uniformly dual or hybrid single-dual duplex edges, respectively, these DNA-NPs are relatively compliant mechanically and are therefore of limited utility for some applications. Further, these algorithms are incapable of handling DNA-NP edge designs composed of more than two duplexes, which are needed to enhance DNA-NP mechanical stiffness. As an alternative, here we introduce the scaffolded DNA origami sequence design algorithm TALOS, which is a generalized procedure for the fully automated design of wireframe 3D polyhedra composed of edges of any cross section with an even number of duplexes, and apply it to DNA-NPs composed uniformly of single honeycomb edges. We also introduce a multiway vertex design that enables the fabrication of DNA-NPs with arbitrary edge lengths and vertex angles and apply it to synthesize a highly asymmetric origami object. Sequence designs are demonstrated to fold robustly into target DNA-NP shapes with high folding efficiency and structural fidelity that is verified using single particle cryo-electron microscopy and 3D reconstruction. In order to test its generality, we apply TALOS to design an  in silico library of over 200 DNA-NPs of distinct symmetries and sizes, and for broad impact, we also provide the software as open source for the generation of custom NP designs.

    View details for PubMedID 30605605

  • NANOSCALE ELUCIDATION OF THE INVASION APPARATUS OF APICOMPLEXAN PARASITES Segev-Zarko, L., Sun, S. Y., Dahlberg, P. D., Pelt, D., Chen, J., Schmid, M. F., Galaz-Montoya, J., Moerner, W. E., Larabell, C., Sethian, J., Chiu, W., Boothroyd, J. AMER SOC TROP MED & HYGIENE. 2019: 620
  • Photo-controlled release of paclitaxel and model drugs from RNA pyramids. Nano research Xu, C., Li, H., Zhang, K., Binzel, D. W., Yin, H., Chiu, W., Guo, P. 2019; 12 (1): 41–48

    Abstract

    Stimuli-responsive release of drugs from a nanocarrier in spatial-, temporal-, and dosage-controlled fashions is of great interest in the pharmaceutical industry. Paclitaxel is one of the most effective and popular chemotherapeutic drugs against a number of cancers such as metastatic or nonmetastatic breast cancer, non-small cell lung cancer, refractory ovarian cancer, AIDS-related Kaposi's sarcoma, and head and neck cancers. Here, by taking the advantage of RNA nanotechnology in biomedical and material science, we developed a three-dimensional pyramid-shaped RNA nanocage for a photocontrolled release of cargo, using paclitaxel as a model drug. The light-triggered release of paclitaxel or fluorophore Cy5 was achieved by incorporation of photocleavable spacers into the RNA nanoparticles. Upon irradiation with ultraviolet light, cargos were rapidly released (within 5 min). In vitro treatment of breast cancer cells with the RNA nanoparticles harboring photocleavable paclitaxel showed higher cytotoxicity as compared to RNA nanoparticles without the photocleavable spacer. The methodology provides proof of concept for the application of the light-triggered controlled release of drugs from RNA nanocages.

    View details for DOI 10.1007/s12274-018-2174-x

    View details for PubMedID 31258852

    View details for PubMedCentralID PMC6599617

  • Structures of TRPV2 in distinct conformations provide insight into role of the pore turret NATURE STRUCTURAL & MOLECULAR BIOLOGY Dosey, T. L., Wang, Z., Fan, G., Zhang, Z., Serysheva, I. I., Chiu, W., Wensel, T. G. 2019; 26 (1): 40-+

    Abstract

    Cation channels of the transient receptor potential (TRP) family serve important physiological roles by opening in response to diverse intra- and extracellular stimuli that regulate their lower or upper gates. Despite extensive studies, the mechanism coupling these gates has remained obscure. Previous structures have failed to resolve extracellular loops, known in the TRPV subfamily as 'pore turrets', which are proximal to the upper gates. We established the importance of the pore turret through activity assays and by solving structures of rat TRPV2, both with and without an intact turret at resolutions of 4.0 Å and 3.6 Å, respectively. These structures resolve the full-length pore turret and reveal fully open and partially open states of TRPV2, both with unoccupied vanilloid pockets. Our results suggest a mechanism by which physiological signals, such as lipid binding, can regulate the lower gate and couple to the upper gate through a pore-turret-facilitated mechanism.

    View details for DOI 10.1038/s41594-018-0168-8

    View details for Web of Science ID 000454902900006

    View details for PubMedID 30598551

    View details for PubMedCentralID PMC6458597

  • Assessment of structural features in Cryo-EM density maps using SSE and side chain Z-scores JOURNAL OF STRUCTURAL BIOLOGY Pintilie, G., Chiu, W. 2018; 204 (3): 564–71

    Abstract

    We introduce a new method for assessing resolvability of structural features in density maps from Cryo-Electron Microscopy (Cryo-EM) using fitted or derived models. It calculates Z-scores for secondary structure elements (SSEs) and side chains. Z-scores capture how much larger the cross-correlation score (CCS) is for atoms in such features at their placed locations compared to the CCS at displaced positions. Z-scores are larger when the structural features are well-resolved, as confirmed by visual analysis. This method was applied to all 66 maps submitted to the 2015/2016 EMDB map challenge. For each map, the fitted model provided by the map committee was used in this assessment. The average Z-scores for each map and fitted model correlate moderately well with reported map resolutions (r2 = 0.45 for SSE Z-scores and r2 = 0.56 for side chain Z-scores). Rankings of the submitted maps based on average Z-scores seem to more closely agree with visual analysis. Z-scores can also be used to pinpoint which parts of a model are well-resolved in a map, and which parts of the model may need further fitting or refinement to make the model better match the density.

    View details for DOI 10.1016/j.jsb.2018.08.015

    View details for Web of Science ID 000454373000022

    View details for PubMedID 30144506

    View details for PubMedCentralID PMC6525962

  • Comparing cryo-EM structures JOURNAL OF STRUCTURAL BIOLOGY Lawson, C. L., Chiu, W. 2018; 204 (3): 523–26

    View details for DOI 10.1016/j.jsb.2018.10.004

    View details for Web of Science ID 000454373000018

    View details for PubMedID 30321594

    View details for PubMedCentralID PMC6464812

  • Structure of Calcarisporiella thermophila Hsp104 Disaggregase that Antagonizes Diverse Proteotoxic Misfolding Events. Structure (London, England : 1993) Michalska, K., Zhang, K., March, Z. M., Hatzos-Skintges, C., Pintilie, G., Bigelow, L., Castellano, L. M., Miles, L. J., Jackrel, M. E., Chuang, E., Jedrzejczak, R., Shorter, J., Chiu, W., Joachimiak, A. 2018

    Abstract

    Hsp104 is an AAA+ protein disaggregase with powerful amyloid-remodeling activity. All nonmetazoan eukaryotes express Hsp104 while eubacteria express an Hsp104 ortholog, ClpB. However, most studies have focused on Hsp104 from Saccharomyces cerevisiae and ClpB orthologs from two eubacterial species. Thus, the natural spectrum of Hsp104/ClpB molecular architectures and protein-remodeling activities remains largely unexplored. Here, we report two structures of Hsp104 from the thermophilic fungus Calcarisporiella thermophila (CtHsp104), a 2.70A crystal structure and 4.0A cryo-electron microscopystructure. Both structures reveal left-handed, helical assemblies with all domains clearly resolved. We thus provide the highest resolution and most complete view of Hsp104 hexamers to date. We also establish that CtHsp104 antagonizes several toxic protein-misfolding events invivo where S. cerevisiae Hsp104 is ineffective, including rescue of TDP-43, polyglutamine, and alpha-synuclein toxicity. We suggest that natural Hsp104 variation is an invaluable, untapped resource for illuminating therapeutic disaggregases for fatal neurodegenerative diseases.

    View details for PubMedID 30595457

  • Programming molecular topologies from single-stranded nucleic acids. Nature communications Qi, X., Zhang, F., Su, Z., Jiang, S., Han, D., Ding, B., Liu, Y., Chiu, W., Yin, P., Yan, H. 2018; 9 (1): 4579

    Abstract

    Molecular knots represent one of the most extraordinary topological structures in biological polymers. Creating highly knotted nanostructures with well-defined and sophisticated geometries and topologies remains challenging. Here, we demonstrate a general strategy to design and construct highly knotted nucleic acid nanostructures, each weaved from a single-stranded DNA or RNA chain by hierarchical folding in a prescribed order. Sets of DNA and RNA knots of two- or three-dimensional shapes have been designed and constructed (ranging from 1700 to 7500 nucleotides), and they exhibit complex topological features, with high crossing numbers (from 9 up to 57). These single-stranded DNA/RNA knots can be replicated and amplified enzymatically in vitro andin vivo. This work establishes a general platform for constructing nucleic acid nanostructures with complex molecular topologies.

    View details for PubMedID 30389935

  • The first single particle analysis Map Challenge: A summary of the assessments JOURNAL OF STRUCTURAL BIOLOGY Heymann, J., Marabini, R., Kazemi, M., Sorzano, C. S., Holmdahl, M., Mendez, J. H., Stagg, S. M., Jonic, S., Palovcak, E., Armache, J., Zhao, J., Cheng, Y., Pintilie, G., Chiu, W., Patwardhan, A., Carazo, J. 2018; 204 (2): 291–300

    Abstract

    The recent successes of cryo-electron microscopy fostered great expectation of solving many new and previously recalcitrant biomolecular structures. However, it also brings with it the danger of compromising the validity of the outcomes if not done properly. The Map Challenge is a first step in assessing the state of the art and to shape future developments in data processing. The organizers presented seven cases for single particle reconstruction, and 27 members of the community responded with 66 submissions. Seven groups analyzed these submissions, resulting in several assessment reports, summarized here. We devised a range of analyses to evaluate the submitted maps, including visual impressions, Fourier shell correlation, pairwise similarity and interpretation through modeling. Unfortunately, we did not find strong trends. We ascribe this to the complexity of the challenge, dealing with multiple cases, software packages and processing approaches. This puts the user in the spotlight, where his/her choices becomes the determinant of map quality. The future focus should therefore be on promulgating best practices and encapsulating these in the software. Such practices include adherence to validation principles, most notably the processing of independent sets, proper resolution-limited alignment, appropriate masking and map sharpening. We consider the Map Challenge to be a highly valuable exercise that should be repeated frequently or on an ongoing basis.

    View details for DOI 10.1016/j.jsb.2018.08.010

    View details for Web of Science ID 000447226400018

    View details for PubMedID 30114512

    View details for PubMedCentralID PMC6205511

  • Evaluation system and web infrastructure for the second cryo-EM model challenge JOURNAL OF STRUCTURAL BIOLOGY Kryshtafovych, A., Adams, P. D., Lawson, C. L., Chiu, W. 2018; 204 (1): 96–108

    Abstract

    An evaluation system and a web infrastructure were developed for the second cryo-EM model challenge. The evaluation system includes tools to validate stereo-chemical plausibility of submitted models, check their fit to the corresponding density maps, estimate their overall and per-residue accuracy, and assess their similarity to reference cryo-EM or X-ray structures as well as other models submitted in this challenge. The web infrastructure provides a convenient interface for analyzing models at different levels of detail. It includes interactively sortable tables of evaluation scores for different subsets of models and different sublevels of structure organization, and a suite of visualization tools facilitating model analysis. The results are publicly accessible at http://model-compare.emdatabank.org.

    View details for DOI 10.1016/j.jsb.2018.07.006

    View details for Web of Science ID 000444661500013

    View details for PubMedID 30017700

    View details for PubMedCentralID PMC6205695

  • Distribution of evaluation scores for the models submitted to the second cryo-EM model challenge DATA IN BRIEF Kryshtafovych, A., Monastyrskyy, B., Adams, P. D., Lawson, C. L., Chiu, W. 2018; 20: 1629–38

    Abstract

    142 protein structure models were submitted to second Cryo-EM model challenge (2015-2016). Accuracy of the models was evaluated with 54 evaluation scores. Results of the descriptive statistical analysis of the scores are provided in this article.

    View details for DOI 10.1016/j.dib.2018.08.214

    View details for Web of Science ID 000450242200239

    View details for PubMedID 30263915

    View details for PubMedCentralID PMC6157618

  • Machining protein microcrystals for structure determination by electron diffraction PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Duyvesteyn, H. E., Kotecha, A., Ginn, H. M., Hecksel, C. W., Beale, E. V., de Haas, F., Evans, G., Zhang, P., Chiu, W., Stuart, D. I. 2018; 115 (38): 9569–73

    Abstract

    We demonstrate that ion-beam milling of frozen, hydrated protein crystals to thin lamella preserves the crystal lattice to near-atomic resolution. This provides a vehicle for protein structure determination, bridging the crystal size gap between the nanometer scale of conventional electron diffraction and micron scale of synchrotron microfocus beamlines. The demonstration that atomic information can be retained suggests that milling could provide such detail on sections cut from vitrified cells.

    View details for DOI 10.1073/pnas.1809978115

    View details for Web of Science ID 000447224900066

    View details for PubMedID 30171169

    View details for PubMedCentralID PMC6156647

  • Neutralizing Antibodies Inhibit Chikungunya Virus Budding at the Plasma Membrane CELL HOST & MICROBE Jin, J., Galaz-Montoya, J. G., Sherman, M. B., Sun, S. Y., Goldsmith, C. S., O'Toole, E. T., Ackerman, L., Carlson, L., Weaver, S. C., Chiu, W., Simmons, G. 2018; 24 (3): 417-+

    Abstract

    Neutralizing antibodies (NAbs) are traditionally thought to inhibit virus infection by preventing virion entry into target cells. In addition, antibodies can engage Fc receptors (FcRs) on immune cells to activate antiviral responses. We describe a mechanism by which NAbs inhibit chikungunya virus (CHIKV), the most common alphavirus infecting humans, by preventing virus budding from infected human cells and activating IgG-specific Fcγ receptors. NAbs bind to CHIKV glycoproteins on the infected cell surface and induce glycoprotein coalescence, preventing budding of nascent virions and leaving structurally heterogeneous nucleocapsids arrested in the cytosol. Furthermore, NAbs induce clustering of CHIKV replication spherules at sites of budding blockage. Functionally, these densely packed glycoprotein-NAb complexes on infected cells activate Fcγ receptors, inducing a strong, antibody-dependent, cell-mediated cytotoxicity response from immune effector cells. Our findings describe a triply functional antiviral pathway for NAbs that might be broadly applicable across virus-host systems, suggesting avenues for therapeutic innovation through antibody design.

    View details for PubMedID 30146390

    View details for PubMedCentralID PMC6137268

  • Evaluation of models in the 2016 Cryo-EM Model Challenge Kryshtafovych, A., Lawson, C. L., Chiu, W. INT UNION CRYSTALLOGRAPHY. 2018: A123
  • Flagellum couples cell shape to motility in Trypanosoma brucei. Proceedings of the National Academy of Sciences of the United States of America Sun, S. Y., Kaelber, J. T., Chen, M., Dong, X., Nematbakhsh, Y., Shi, J., Dougherty, M., Lim, C. T., Schmid, M. F., Chiu, W., He, C. Y. 2018; 115 (26): E5916–E5925

    Abstract

    In the unicellular parasite Trypanosoma brucei, the causative agent of human African sleeping sickness, complex swimming behavior is driven by a flagellum laterally attached to the long and slender cell body. Using microfluidic assays, we demonstrated that T. brucei can penetrate through an orifice smaller than its maximum diameter. Efficient motility and penetration depend on active flagellar beating. To understand how active beating of the flagellum affects the cell body, we genetically engineered T. brucei to produce anucleate cytoplasts (zoids and minis) with different flagellar attachment configurations and different swimming behaviors. We used cryo-electron tomography (cryo-ET) to visualize zoids and minis vitrified in different motility states. We showed that flagellar wave patterns reflective of their motility states are coupled to cytoskeleton deformation. Based on these observations, we propose a mechanism for how flagellum beating can deform the cell body via a flexible connection between the flagellar axoneme and the cell body. This mechanism may be critical for T. brucei to disseminate in its host through size-limiting barriers.

    View details for PubMedID 29891682

  • Novel Insect-Specific Eilat Virus-Based Chimeric Vaccine Candidates Provide Durable, Mono- and Multivalent, Single-Dose Protection against Lethal Alphavirus Challenge. Journal of virology Erasmus, J. H., Seymour, R. L., Kaelber, J. T., Kim, D. Y., Leal, G., Sherman, M. B., Frolov, I., Chiu, W., Weaver, S. C., Nasar, F. 2018; 92 (4)

    Abstract

    Most alphaviruses are mosquito borne and exhibit a broad host range, infecting many different vertebrates, including birds, rodents, equids, humans, and nonhuman primates. Recently, a host-restricted, mosquito-borne alphavirus, Eilat virus (EILV), was described with an inability to infect vertebrate cells based on defective attachment and/or entry, as well as a lack of genomic RNA replication. We investigated the utilization of EILV recombinant technology as a vaccine platform against eastern (EEEV) and Venezuelan equine encephalitis viruses (VEEV), two important pathogens of humans and domesticated animals. EILV chimeras containing structural proteins of EEEV or VEEV were engineered and successfully rescued in Aedes albopictus cells. Cryo-electron microscopy reconstructions at 8 and 11 Å of EILV/VEEV and EILV/EEEV, respectively, showed virion and glycoprotein spike structures similar to those of VEEV-TC83 and other alphaviruses. The chimeras were unable to replicate in vertebrate cell lines or in brains of newborn mice when injected intracranially. Histopathologic examinations of the brain tissues showed no evidence of pathological lesions and were indistinguishable from those of mock-infected animals. A single-dose immunization of either monovalent or multivalent EILV chimera(s) generated neutralizing antibody responses and protected animals against lethal challenge 70 days later. Lastly, a single dose of monovalent EILV chimeras generated protective responses as early as day 1 postvaccination and partial or complete protection by day 6. These data demonstrate the safety, immunogenicity, and efficacy of novel insect-specific EILV-based chimeras as potential EEEV and VEEV vaccines.IMPORTANCE Mostly in the last decade, insect-specific viruses have been discovered in several arbovirus families. However, most of these viruses are not well studied and largely have been ignored. We explored the use of the mosquito-specific alphavirus EILV as an alphavirus vaccine platform in well-established disease models for eastern (EEE) and Venezuelan equine encephalitis (VEE). EILV-based chimeras replicated to high titers in a mosquito cell line yet retained their host range restriction in vertebrates both in vitro and in vivo In addition, the chimeras generated immune responses that were higher than those of other human and/or equine vaccines. These findings indicate the feasibility of producing a safe, efficacious, mono- or multivalent vaccine against the encephalitic alphaviruses VEEV and EEEV. Lastly, these data demonstrate how host-restricted, insect-specific viruses can be engineered to develop vaccines against related pathogenic arboviruses that cause severe disease in humans and domesticated animals.

    View details for DOI 10.1128/JVI.01274-17

    View details for PubMedID 29187545

    View details for PubMedCentralID PMC5790933

  • Structure of the 30 kDa HIV-1 RNA Dimerization Signal by a Hybrid Cryo-EM, NMR, and Molecular Dynamics Approach Structure of the 30 kDa HIV-1 RNA Dimerization Signal by a Hybrid Cryo-EM, NMR, and Molecular Dynamics Approach Zhang, K., keane, S. C., Su, Z., robalieva, R., Chen, M., Sciandra, C. A., Marchant, J., Heng, X., Schmid, M. F., Case, D. A., Ludtke, S. J., Summers, M. F., Chiu, W. 2018; 26 (3): 490-498

    Abstract

    Cryoelectron microscopy (cryo-EM) and nuclear magnetic resonance (NMR) spectroscopy are routinely used to determine structures of macromolecules with molecular weights over 65 and under 25 kDa, respectively. We combined these techniques to study a 30 kDa HIV-1 dimer initiation site RNA ([DIS]2; 47 nt/strand). A 9 Å cryo-EM map clearly shows major groove features of the double helix and a right-handed superhelical twist. Simulated cryo-EM maps generated from time-averaged molecular dynamics trajectories (10 ns) exhibited levels of detail similar to those in the experimental maps, suggesting internal structural flexibility limits the cryo-EM resolution. Simultaneous inclusion of the cryo-EM map and 2H-edited NMR-derived distance restraints during structure refinement generates a structure consistent with both datasets and supporting a flipped-out base within a conserved purine-rich bulge. Our findings demonstrate the power of combining global and local structural information from these techniques for structure determination of modest-sized RNAs.

    View details for DOI 10.1016/j.str.2018.01.001

    View details for PubMedCentralID PMC5842133

  • Electron Cryo-microscopy Structure of Ebola Virus Nucleoprotein Reveals a Mechanism for Nucleocapsid-like Assembly Cell Su, Z., et al 2018; 172 (5): 966-978

    Abstract

    Ebola virus nucleoprotein (eNP) assembles into higher-ordered structures that form the viral nucleocapsid (NC) and serve as the scaffold for viral RNA synthesis. However, molecular insights into the NC assembly process are lacking. Using a hybrid approach, we characterized the NC-like assembly of eNP, identified novel regulatory elements, and described how these elements impact function. We generated a three-dimensional structure of the eNP NC-like assembly at 5.8 Å using electron cryo-microscopy and identified a new regulatory role for eNP helices α22-α23. Biochemical, biophysical, and mutational analyses revealed that inter-eNP contacts within α22-α23 are critical for viral NC assembly and regulate viral RNA synthesis. These observations suggest that the N terminus and α22-α23 of eNP function as context-dependent regulatory modules (CDRMs). Our current study provides a framework for a structural mechanism for NC-like assembly and a new therapeutic target.

    View details for DOI 10.1016/j.cell.2018.02.009

    View details for PubMedCentralID PMC5973842

  • Purification of AcrAB-TolC Multidrug Efflux Pump for Cryo-EM Analysis. Methods in molecular biology (Clifton, N.J.) Du, D., Wang, Z., Chiu, W., Luisi, B. F. 2018; 1700: 71–81

    Abstract

    The cell envelope of Gram-negative bacteria comprises an outer membrane, a cytoplasmic inner membrane, and an interstitial space. The tripartite multidrug transporter AcrAB-TolC, which uses proton electrochemical gradients to vectorially drive the efflux of drugs from the cell, spans this envelope. We describe here details of the methods used to prepare the recombinant tripartite assembly for high-resolution structure determination by cryo-EM.

    View details for DOI 10.1007/978-1-4939-7454-2_5

    View details for PubMedID 29177826

  • Editorial overview: Cryo Electron Microscopy: Exciting advances in CryoEM Herald a new era in structural biology CURRENT OPINION IN STRUCTURAL BIOLOGY Chiu, W., Downing, K. H. 2017; 46: IV-VIII

    View details for Web of Science ID 000416299600001

    View details for PubMedID 28801059

    View details for PubMedCentralID PMC5683930

  • GENFIRE: A generalized Fourier iterative reconstruction algorithm for high-resolution 3D imaging SCIENTIFIC REPORTS Pryor, A., Yang, Y., Rana, A., Gallagher-Jones, M., Zhou, J., Lo, Y., Melinte, G., Chiu, W., Rodriguez, J. A., Miao, J. 2017; 7: 10409

    Abstract

    Tomography has made a radical impact on diverse fields ranging from the study of 3D atomic arrangements in matter to the study of human health in medicine. Despite its very diverse applications, the core of tomography remains the same, that is, a mathematical method must be implemented to reconstruct the 3D structure of an object from a number of 2D projections. Here, we present the mathematical implementation of a tomographic algorithm, termed GENeralized Fourier Iterative REconstruction (GENFIRE), for high-resolution 3D reconstruction from a limited number of 2D projections. GENFIRE first assembles a 3D Fourier grid with oversampling and then iterates between real and reciprocal space to search for a global solution that is concurrently consistent with the measured data and general physical constraints. The algorithm requires minimal human intervention and also incorporates angular refinement to reduce the tilt angle error. We demonstrate that GENFIRE can produce superior results relative to several other popular tomographic reconstruction techniques through numerical simulations and by experimentally reconstructing the 3D structure of a porous material and a frozen-hydrated marine cyanobacterium. Equipped with a graphical user interface, GENFIRE is freely available from our website and is expected to find broad applications across different disciplines.

    View details for DOI 10.1038/s41598-017-09847-1

    View details for Web of Science ID 000409308800001

    View details for PubMedID 28874736

    View details for PubMedCentralID PMC5585178

  • SuRVoS: Super-Region Volume Segmentation workbench JOURNAL OF STRUCTURAL BIOLOGY Luengo, I., Darrow, M. C., Spink, M. C., Sun, Y., Dai, W., He, C. Y., Chiu, W., Pridmore, T., Ashton, A. W., Duke, E. M., Basham, M., French, A. P. 2017; 198 (1): 43-53

    Abstract

    Segmentation of biological volumes is a crucial step needed to fully analyse their scientific content. Not having access to convenient tools with which to segment or annotate the data means many biological volumes remain under-utilised. Automatic segmentation of biological volumes is still a very challenging research field, and current methods usually require a large amount of manually-produced training data to deliver a high-quality segmentation. However, the complex appearance of cellular features and the high variance from one sample to another, along with the time-consuming work of manually labelling complete volumes, makes the required training data very scarce or non-existent. Thus, fully automatic approaches are often infeasible for many practical applications. With the aim of unifying the segmentation power of automatic approaches with the user expertise and ability to manually annotate biological samples, we present a new workbench named SuRVoS (Super-Region Volume Segmentation). Within this software, a volume to be segmented is first partitioned into hierarchical segmentation layers (named Super-Regions) and is then interactively segmented with the user's knowledge input in the form of training annotations. SuRVoS first learns from and then extends user inputs to the rest of the volume, while using Super-Regions for quicker and easier segmentation than when using a voxel grid. These benefits are especially noticeable on noisy, low-dose, biological datasets.

    View details for DOI 10.1016/j.jsb.2017.02.007

    View details for Web of Science ID 000400318400007

    View details for PubMedID 28246039

    View details for PubMedCentralID PMC5405849

  • Responses to 'Atomic resolution': a badly abused term in structural biology ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY Chiu, W., Holton, J., Langan, P., Sauter, N. K., Schlichting, I., Terwilliger, T., Martin, J. L., Read, R. J., Wakatsuki, S. 2017; 73: 381-383

    View details for DOI 10.1107/S205979831700417X

    View details for Web of Science ID 000398536800010

    View details for PubMedID 28375150

  • An allosteric transport mechanism for the AcrAB-TolC multidrug efflux pump ELIFE Wang, Z., Fan, G., Hryc, C. F., Blaza, J. N., Serysheva, I. I., Schmid, M. F., Chiu, W., Luisi, B. F., Du, D. 2017; 6

    Abstract

    Bacterial efflux pumps confer multidrug resistance by transporting diverse antibiotics from the cell. In Gram-negative bacteria, some of these pumps form multi-protein assemblies that span the cell envelope. Here, we report the near-atomic resolution cryoEM structures of the Escherichia coli AcrAB-TolC multidrug efflux pump in resting and drug transport states, revealing a quaternary structural switch that allosterically couples and synchronizes initial ligand binding with channel opening. Within the transport-activated state, the channel remains open even though the pump cycles through three distinct conformations. Collectively, our data provide a dynamic mechanism for the assembly and operation of the AcrAB-TolC pump.

    View details for DOI 10.7554/eLife.24905

    View details for PubMedID 28355133

  • Visualizing Adsorption of Cyanophage P-SSP7 onto Marine Prochlorococcus SCIENTIFIC REPORTS Murata, K., Zhang, Q., Galaz-Montoya, J. G., Fu, C., Coleman, M. L., Osburne, M. S., Schmid, M. F., Sullivan, M. B., Chisholm, S. W., Chiu, W. 2017; 7

    Abstract

    Marine cyanobacteria perform roughly a quarter of global carbon fixation, and cyanophages that infect them liberate some of this carbon during infection and cell lysis. Studies of the cyanobacterium Prochlorococcus MED4 and its associated cyanophage P-SSP7 have revealed complex gene expression dynamics once infection has begun, but the initial cyanophage-host interactions remain poorly understood. Here, we used single particle cryo-electron tomography (cryo-ET) to investigate cyanophage-host interactions in this model system, based on 170 cyanophage-to-host adsorption events. Subtomogram classification and averaging revealed three main conformations characterized by different angles between the phage tail and the cell surface. Namely, phage tails were (i) parallel to, (ii) ~45 degrees to, or (iii) perpendicular to the cell surface. Furthermore, different conformations of phage tail fibers correlated with the aforementioned orientations of the tails. We also observed density beyond the tail tip in vertically-oriented phages that had penetrated the cell wall, capturing the final stage of adsorption. Together, our data provide a quantitative characterization of the orientation of phages as they adsorb onto cells, and suggest that cyanophages that abut their cellular targets are only transiently in the "perpendicular" orientation required for successful infection.

    View details for DOI 10.1038/srep44176

    View details for Web of Science ID 000395891400001

    View details for PubMedID 28281671

    View details for PubMedCentralID PMC5345008

  • Subunit conformational variation within individual GroEL oligomers resolved by Cryo-EM Proc Natl Acad Sci U S A Roh, S. H., Hryc, C. F., Jeong, H. H., Fei, H. H., Jakana, J., Lorimer, G. H., Chiu, W. 2017; 114 (31): 8259-8264

    View details for DOI 10.1073/pnas.1704725114

  • A chikungunya fever vaccine utilizing an insect-specific virus platform. Nature medicine Erasmus, J. H., Auguste, A. J., Kaelber, J. T., Luo, H., Rossi, S. L., Fenton, K., Leal, G., Kim, D. Y., Chiu, W., Wang, T., Frolov, I., Nasar, F., Weaver, S. C. 2017; 23 (2): 192–99

    Abstract

    Traditionally, vaccine development involves tradeoffs between immunogenicity and safety. Live-attenuated vaccines typically offer rapid and durable immunity but have reduced safety when compared to inactivated vaccines. In contrast, the inability of inactivated vaccines to replicate enhances safety at the expense of immunogenicity, often necessitating multiple doses and boosters. To overcome these tradeoffs, we developed the insect-specific alphavirus, Eilat virus (EILV), as a vaccine platform. To address the chikungunya fever (CHIKF) pandemic, we used an EILV cDNA clone to design a chimeric virus containing the chikungunya virus (CHIKV) structural proteins. The recombinant EILV/CHIKV was structurally identical at 10 Å to wild-type CHIKV, as determined by single-particle cryo-electron microscopy, and it mimicked the early stages of CHIKV replication in vertebrate cells from attachment and entry to viral RNA delivery. Yet the recombinant virus remained completely defective for productive replication, providing a high degree of safety. A single dose of EILV/CHIKV produced in mosquito cells elicited rapid (within 4 d) and long-lasting (>290 d) neutralizing antibodies that provided complete protection in two different mouse models. In nonhuman primates, EILV/CHIKV elicited rapid and robust immunity that protected against viremia and telemetrically monitored fever. Our EILV platform represents the first structurally native application of an insect-specific virus in preclinical vaccine development and highlights the potential application of such viruses in vaccinology.

    View details for DOI 10.1038/nm.4253

    View details for PubMedID 27991917

    View details for PubMedCentralID PMC5296253

  • Influence of DNA sequence on the structure of minicircles under torsional stress. Nucleic acids research Wang, Q., Irobalieva, R. N., Chiu, W., Schmid, M. F., Fogg, J. M., Zechiedrich, L., Pettitt, B. M. 2017; 45 (13): 7633–42

    Abstract

    The sequence dependence of the conformational distribution of DNA under various levels of torsional stress is an important unsolved problem. Combining theory and coarse-grained simulations shows that the DNA sequence and a structural correlation due to topology constraints of a circle are the main factors that dictate the 3D structure of a 336 bp DNA minicircle under torsional stress. We found that DNA minicircle topoisomers can have multiple bend locations under high torsional stress and that the positions of these sharp bends are determined by the sequence, and by a positive mechanical correlation along the sequence. We showed that simulations and theory are able to provide sequence-specific information about individual DNA minicircles observed by cryo-electron tomography (cryo-ET). We provided a sequence-specific cryo-ET tomogram fitting of DNA minicircles, registering the sequence within the geometric features. Our results indicate that the conformational distribution of minicircles under torsional stress can be designed, which has important implications for using minicircle DNA for gene therapy.

    View details for DOI 10.1093/nar/gkx516

    View details for PubMedID 28609782

    View details for PubMedCentralID PMC5737869

  • Subunit conformational variation within individual GroEL oligomers resolved by Cryo-EM. Proceedings of the National Academy of Sciences of the United States of America Roh, S. H., Hryc, C. F., Jeong, H. H., Fei, X., Jakana, J., Lorimer, G. H., Chiu, W. 2017; 114 (31): 8259–64

    Abstract

    Single-particle electron cryo-microscopy (cryo-EM) is an emerging tool for resolving structures of conformationally heterogeneous particles; however, each structure is derived from an average of many particles with presumed identical conformations. We used a 3.5-Å cryo-EM reconstruction with imposed D7 symmetry to further analyze structural heterogeneity among chemically identical subunits in each GroEL oligomer. Focused classification of the 14 subunits in each oligomer revealed three dominant classes of subunit conformations. Each class resembled a distinct GroEL crystal structure in the Protein Data Bank. The conformational differences stem from the orientations of the apical domain. We mapped each conformation class to its subunit locations within each GroEL oligomer in our dataset. The spatial distributions of each conformation class differed among oligomers, and most oligomers contained 10-12 subunits of the three dominant conformation classes. Adjacent subunits were found to more likely assume the same conformation class, suggesting correlation among subunits in the oligomer. This study demonstrates the utility of cryo-EM in revealing structure dynamics within a single protein oligomer.

    View details for DOI 10.1073/pnas.1704725114

    View details for PubMedID 28710336

    View details for PubMedCentralID PMC5547627

  • Electron Cryomicroscopy of Viruses at Near-Atomic Resolutions. Annual review of virology Kaelber, J. T., Hryc, C. F., Chiu, W. 2017; 4 (1): 287–308

    Abstract

    Recently, dozens of virus structures have been solved to resolutions between 2.5 and 5.0 Å by means of electron cryomicroscopy. With these structures we are now firmly within the "atomic age" of electron cryomicroscopy, as these studies can reveal atomic details of protein and nucleic acid topology and interactions between specific residues. This improvement in resolution has been the result of direct electron detectors and image processing advances. Although enforcing symmetry facilitates reaching near-atomic resolution with fewer particle images, it unfortunately obscures some biologically interesting components of a virus. New approaches on relaxing symmetry and exploring structure dynamics and heterogeneity of viral assemblies have revealed important insights into genome packaging, virion assembly, cell entry, and other stages of the viral life cycle. In the future, novel methods will be required to reveal yet-unknown structural conformations of viruses, relevant to their biological activities. Ultimately, these results hold the promise of answering many unresolved questions linking structural diversity of viruses to their biological functions.

    View details for DOI 10.1146/annurev-virology-101416-041921

    View details for PubMedID 28715974

  • Structural and Functional Impacts of ER Coactivator Sequential Recruitment. Molecular cell Yi, P., Wang, Z., Feng, Q., Chou, C. K., Pintilie, G. D., Shen, H., Foulds, C. E., Fan, G., Serysheva, I., Ludtke, S. J., Schmid, M. F., Hung, M. C., Chiu, W., O'Malley, B. W. 2017; 67 (5): 733–43.e4

    Abstract

    Nuclear receptors recruit multiple coactivators sequentially to activate transcription. This "ordered" recruitment allows different coactivator activities to engage the nuclear receptor complex at different steps of transcription. Estrogen receptor (ER) recruits steroid receptor coactivator-3 (SRC-3) primary coactivator and secondary coactivators, p300/CBP and CARM1. CARM1 recruitment lags behind the binding of SRC-3 and p300 to ER. Combining cryo-electron microscopy (cryo-EM) structure analysis and biochemical approaches, we demonstrate that there is a close crosstalk between early- and late-recruited coactivators. The sequential recruitment of CARM1 not only adds a protein arginine methyltransferase activity to the ER-coactivator complex, it also alters the structural organization of the pre-existing ERE/ERα/SRC-3/p300 complex. It induces a p300 conformational change and significantly increases p300 HAT activity on histone H3K18 residues, which, in turn, promotes CARM1 methylation activity on H3R17 residues to enhance transcriptional activity. This study reveals a structural role for a coactivator sequential recruitment and biochemical process in ER-mediated transcription.

    View details for DOI 10.1016/j.molcel.2017.07.026

    View details for PubMedID 28844863

    View details for PubMedCentralID PMC5657569

  • Convolutional neural networks for automated annotation of cellular cryo-electron tomograms. Nature methods Chen, M., Dai, W., Sun, S. Y., Jonasch, D., He, C. Y., Schmid, M. F., Chiu, W., Ludtke, S. J. 2017; 14 (10): 983–85

    Abstract

    Cellular electron cryotomography offers researchers the ability to observe macromolecules frozen in action in situ, but a primary challenge with this technique is identifying molecular components within the crowded cellular environment. We introduce a method that uses neural networks to dramatically reduce the time and human effort required for subcellular annotation and feature extraction. Subsequent subtomogram classification and averaging yield in situ structures of molecular components of interest. The method is available in the EMAN2.2 software package.

    View details for DOI 10.1038/nmeth.4405

    View details for PubMedID 28846087

    View details for PubMedCentralID PMC5623144

  • Programmable Supra-Assembly of a DNA Surface Adapter for Tunable Chiral Directional Self-Assembly of Gold Nanorods. Angewandte Chemie (International ed. in English) Lan, X., Su, Z., Zhou, Y., Meyer, T., Ke, Y., Wang, Q., Chiu, W., Liu, N., Zou, S., Yan, H., Liu, Y. 2017; 56 (46): 14632–36

    Abstract

    An important challenge in molecular assembly and hierarchical molecular engineering is to control and program the directional self-assembly into chiral structures. Here, we present a versatile DNA surface adapter that can programmably self-assemble into various chiral supramolecular architectures, thereby regulating the chiral directional "bonding" of gold nanorods decorated by the surface adapter. Distinct optical chirality relevant to the ensemble conformation is demonstrated from the assembled novel stair-like and coil-like gold nanorod chiral metastructures, which is strongly affected by the spatial arrangement of neighboring nanorod pair. Our strategy provides new avenues for fabrication of tunable optical metamaterials by manipulating the directional self-assembly of nanoparticles using programmable surface adapters.

    View details for DOI 10.1002/anie.201709775

    View details for PubMedID 28971555

    View details for PubMedCentralID PMC5851444

  • Fabrication of RNA 3D Nanoprisms for Loading and Protection of Small RNAs and Model Drugs ADVANCED MATERIALS Khisamutdinov, E. F., Jasinski, D. L., Li, H., Zhang, K., Chiu, W., Guo, P. 2016; 28 (45): 10079–87

    Abstract

    Constructing containers with defined shape and size to load and protect therapeutics and subsequently control their release in the human body has long been a dream. The fabrication of 3D RNA prisms, characterized by atomic force microscopy, cryo-electron microscopy, dynamic light scattering, and polyacrylamide gel electrophoresis, is reported for the loading and protection of small molecules, proteins, small RNA molecules, and their controlled release.

    View details for PubMedID 27758001

    View details for PubMedCentralID PMC5224701

  • Control of the structural landscape and neuronal proteotoxicity of mutant Huntingtin by domains flanking the polyQ tract. eLife Shen, K., Calamini, B., Fauerbach, J. A., Ma, B., Shahmoradian, S. H., Serrano Lachapel, I. L., Chiu, W., Lo, D. C., Frydman, J. 2016; 5

    Abstract

    Many neurodegenerative diseases are linked to amyloid aggregation. In Huntington's disease (HD), neurotoxicity correlates with an increased aggregation propensity of a polyglutamine (polyQ) expansion in exon 1 of mutant huntingtin protein (mHtt). Here we establish how the domains flanking the polyQ tract shape the mHtt conformational landscape in vitro and in neurons. In vitro, the flanking domains have opposing effects on the conformation and stabilities of oligomers and amyloid fibrils. The N-terminal N17 promotes amyloid fibril formation, while the C-terminal Proline Rich Domain destabilizes fibrils and enhances oligomer formation. However, in neurons both domains act synergistically to engage protective chaperone and degradation pathways promoting mHtt proteostasis. Surprisingly, when proteotoxicity was assessed in rat corticostriatal brain slices, either flanking region alone sufficed to generate a neurotoxic conformation, while the polyQ tract alone exhibited minimal toxicity. Linking mHtt structural properties to its neuronal proteostasis should inform new strategies for neuroprotection in polyQ-expansion diseases.

    View details for DOI 10.7554/eLife.18065

    View details for PubMedID 27751235

    View details for PubMedCentralID PMC5135392

  • Controllable Self-Assembly of RNA Tetrahedrons with Precise Shape and Size for Cancer Targeting ADVANCED MATERIALS Li, H., Zhang, K., Pi, F., Guo, S., Shlyakhtenko, L., Chiu, W., Shu, D., Guo, P. 2016; 28 (34): 7501–7

    Abstract

    RNA tetrahedral nanoparticles with two different sizes are successfully assembled by a one-pot bottom-up approach with high efficiency and thermal stability. The reported design principles can be extended to construct higher-order polyhedral RNA architectures for various applications such as targeted cancer imaging and therapy.

    View details for PubMedID 27322097

    View details for PubMedCentralID PMC5059845

  • DNA NANOTECHNOLOGY Designer nanoscale DNA assemblies programmed from the top down SCIENCE Veneziano, R., Ratanalert, S., Zhang, K., Zhang, F., Yan, H., Chiu, W., Bathe, M. 2016; 352 (6293): 1534

    Abstract

    Scaffolded DNA origami is a versatile means of synthesizing complex molecular architectures. However, the approach is limited by the need to forward-design specific Watson-Crick base pairing manually for any given target structure. Here, we report a general, top-down strategy to design nearly arbitrary DNA architectures autonomously based only on target shape. Objects are represented as closed surfaces rendered as polyhedral networks of parallel DNA duplexes, which enables complete DNA scaffold routing with a spanning tree algorithm. The asymmetric polymerase chain reaction is applied to produce stable, monodisperse assemblies with custom scaffold length and sequence that are verified structurally in three dimensions to be high fidelity by single-particle cryo-electron microscopy. Their long-term stability in serum and low-salt buffer confirms their utility for biological as well as nonbiological applications.

    View details for PubMedID 27229143

    View details for PubMedCentralID PMC5111087

  • Resolution and Probabilistic Models of Components in CryoEM Maps of Mature P22 Bacteriophage BIOPHYSICAL JOURNAL Pintilie, G., Chen, D., Haase-Pettingell, C. A., King, J. A., Chiu, W. 2016; 110 (4): 827-839

    Abstract

    CryoEM continues to produce density maps of larger and more complex assemblies with multiple protein components of mixed symmetries. Resolution is not always uniform throughout a cryoEM map, and it can be useful to estimate the resolution in specific molecular components of a large assembly. In this study, we present procedures to 1) estimate the resolution in subcomponents by gold-standard Fourier shell correlation (FSC); 2) validate modeling procedures, particularly at medium resolutions, which can include loop modeling and flexible fitting; and 3) build probabilistic models that combine high-accuracy priors (such as crystallographic structures) with medium-resolution cryoEM densities. As an example, we apply these methods to new cryoEM maps of the mature bacteriophage P22, reconstructed without imposing icosahedral symmetry. Resolution estimates based on gold-standard FSC show the highest resolution in the coat region (7.6 Å), whereas other components are at slightly lower resolutions: portal (9.2 Å), hub (8.5 Å), tailspike (10.9 Å), and needle (10.5 Å). These differences are indicative of inherent structural heterogeneity and/or reconstruction accuracy in different subcomponents of the map. Probabilistic models for these subcomponents provide new insights, to our knowledge, and structural information when taking into account uncertainty given the limitations of the observed density.

    View details for DOI 10.1016/j.bpj.2015.11.3522

    View details for PubMedID 26743049

  • TRiC subunits enhance BDNF axonal transport and rescue striatal atrophy in Huntington's disease. Proceedings of the National Academy of Sciences of the United States of America Zhao, X., Chen, X. Q., Han, E., Hu, Y., Paik, P., Ding, Z., Overman, J., Lau, A. L., Shahmoradian, S. H., Chiu, W., Thompson, L. M., Wu, C., Mobley, W. C. 2016; 113 (38): E5655–64

    Abstract

    Corticostriatal atrophy is a cardinal manifestation of Huntington's disease (HD). However, the mechanism(s) by which mutant huntingtin (mHTT) protein contributes to the degeneration of the corticostriatal circuit is not well understood. We recreated the corticostriatal circuit in microfluidic chambers, pairing cortical and striatal neurons from the BACHD model of HD and its WT control. There were reduced synaptic connectivity and atrophy of striatal neurons in cultures in which BACHD cortical and striatal neurons were paired. However, these changes were prevented if WT cortical neurons were paired with BACHD striatal neurons; synthesis and release of brain-derived neurotrophic factor (BDNF) from WT cortical axons were responsible. Consistent with these findings, there was a marked reduction in anterograde transport of BDNF in BACHD cortical neurons. Subunits of the cytosolic chaperonin T-complex 1 (TCP-1) ring complex (TRiC or CCT for chaperonin containing TCP-1) have been shown to reduce mHTT levels. Both CCT3 and the apical domain of CCT1 (ApiCCT1) decreased the level of mHTT in BACHD cortical neurons. In cortical axons, they normalized anterograde BDNF transport, restored retrograde BDNF transport, and normalized lysosomal transport. Importantly, treating BACHD cortical neurons with ApiCCT1 prevented BACHD striatal neuronal atrophy by enhancing release of BDNF that subsequently acts through tyrosine receptor kinase B (TrkB) receptor on striatal neurons. Our findings are evidence that TRiC reagent-mediated reductions in mHTT enhanced BDNF delivery to restore the trophic status of BACHD striatal neurons.

    View details for DOI 10.1073/pnas.1603020113

    View details for PubMedID 27601642

    View details for PubMedCentralID PMC5035849

  • Quantifying Variability of Manual Annotation in Cryo-Electron Tomograms. Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada Hecksel, C. W., Darrow, M. C., Dai, W., Galaz-Montoya, J. G., Chin, J. A., Mitchell, P. G., Chen, S., Jakana, J., Schmid, M. F., Chiu, W. 2016; 22 (3): 487–96

    Abstract

    Although acknowledged to be variable and subjective, manual annotation of cryo-electron tomography data is commonly used to answer structural questions and to create a "ground truth" for evaluation of automated segmentation algorithms. Validation of such annotation is lacking, but is critical for understanding the reproducibility of manual annotations. Here, we used voxel-based similarity scores for a variety of specimens, ranging in complexity and segmented by several annotators, to quantify the variation among their annotations. In addition, we have identified procedures for merging annotations to reduce variability, thereby increasing the reliability of manual annotation. Based on our analyses, we find that it is necessary to combine multiple manual annotations to increase the confidence level for answering structural questions. We also make recommendations to guide algorithm development for automated annotation of features of interest.

    View details for DOI 10.1017/S1431927616000799

    View details for PubMedID 27225525

    View details for PubMedCentralID PMC5111626

  • EMDataBank unified data resource for 3DEM. Nucleic acids research Lawson, C. L., Patwardhan, A., Baker, M. L., Hryc, C., Garcia, E. S., Hudson, B. P., Lagerstedt, I., Ludtke, S. J., Pintilie, G., Sala, R., Westbrook, J. D., Berman, H. M., Kleywegt, G. J., Chiu, W. 2016; 44 (D1): D396–403

    Abstract

    Three-dimensional Electron Microscopy (3DEM) has become a key experimental method in structural biology for a broad spectrum of biological specimens from molecules to cells. The EMDataBank project provides a unified portal for deposition, retrieval and analysis of 3DEM density maps, atomic models and associated metadata (emdatabank.org). We provide here an overview of the rapidly growing 3DEM structural data archives, which include maps in EM Data Bank and map-derived models in the Protein Data Bank. In addition, we describe progress and approaches toward development of validation protocols and methods, working with the scientific community, in order to create a validation pipeline for 3DEM data.

    View details for DOI 10.1093/nar/gkv1126

    View details for PubMedID 26578576

    View details for PubMedCentralID PMC4702818

  • Chaperonin TRiC/CCT Modulates the Folding and Activity of Leukemogenic Fusion Oncoprotein AML1-ETO. The Journal of biological chemistry Roh, S. H., Kasembeli, M., Galaz-Montoya, J. G., Trnka, M., Lau, W. C., Burlingame, A., Chiu, W., Tweardy, D. J. 2016; 291 (9): 4732–41

    Abstract

    AML1-ETO is the most common fusion oncoprotein causing acute myeloid leukemia (AML), a disease with a 5-year survival rate of only 24%. AML1-ETO functions as a rogue transcription factor, altering the expression of genes critical for myeloid cell development and differentiation. Currently, there are no specific therapies for AML1-ETO-positive AML. While known for decades to be the translational product of a chimeric gene created by the stable chromosome translocation t(8;21)(q22;q22), it is not known how AML1-ETO achieves its native and functional conformation or whether this process can be targeted for therapeutic benefit. Here, we show that the biosynthesis and folding of the AML1-ETO protein is facilitated by interaction with the essential eukaryotic chaperonin TRiC (or CCT). We demonstrate that a folding intermediate of AML1-ETO binds to TRiC directly, mainly through its β-strand rich, DNA-binding domain (AML-(1-175)), with the assistance of HSP70. Our results suggest that TRiC contributes to AML1-ETO proteostasis through specific interactions between the oncoprotein's DNA-binding domain, which may be targeted for therapeutic benefit.

    View details for DOI 10.1074/jbc.M115.684878

    View details for PubMedID 26706127

    View details for PubMedCentralID PMC4813495

  • The Electron Microscopy eXchange (EMX) initiative. Journal of structural biology Marabini, R., Ludtke, S. J., Murray, S. C., Chiu, W., de la Rosa-Trevín, J. M., Patwardhan, A., Heymann, J. B., Carazo, J. M. 2016; 194 (2): 156–63

    Abstract

    Three-dimensional electron microscopy (3DEM) of ice-embedded samples allows the structural analysis of large biological macromolecules close to their native state. Different techniques have been developed during the last forty years to process cryo-electron microscopy (cryo-EM) data. Not surprisingly, success in analysis and interpretation is highly correlated with the continuous development of image processing packages. The field has matured to the point where further progress in data and methods sharing depends on an agreement between the packages on how to describe common image processing tasks. Such standardization will facilitate the use of software as well as seamless collaboration, allowing the sharing of rich information between different platforms. Our aim here is to describe the Electron Microscopy eXchange (EMX) initiative, launched at the 2012 Instruct Image Processing Center Developer Workshop, with the intention of developing a first set of standard conventions for the interchange of information for single-particle analysis (EMX version 1.0). These conventions cover the specification of the metadata for micrograph and particle images, including contrast transfer function (CTF) parameters and particle orientations. EMX v1.0 has already been implemented in the Bsoft, EMAN, Xmipp and Scipion image processing packages. It has been and will be used in the CTF and EMDataBank Validation Challenges respectively. It is also being used in EMPIAR, the Electron Microscopy Pilot Image Archive, which stores raw image data related to the 3DEM reconstructions in EMDB.

    View details for DOI 10.1016/j.jsb.2016.02.008

    View details for PubMedID 26873784

    View details for PubMedCentralID PMC5093775

  • Alignment algorithms and per-particle CTF correction for single particle cryo-electron tomography. Journal of structural biology Galaz-Montoya, J. G., Hecksel, C. W., Baldwin, P. R., Wang, E., Weaver, S. C., Schmid, M. F., Ludtke, S. J., Chiu, W. 2016; 194 (3): 383–94

    Abstract

    Single particle cryo-electron tomography (cryoSPT) extracts features from cryo-electron tomograms, followed by 3D classification, alignment and averaging to generate improved 3D density maps of such features. Robust methods to correct for the contrast transfer function (CTF) of the electron microscope are necessary for cryoSPT to reach its resolution potential. Many factors can make CTF correction for cryoSPT challenging, such as lack of eucentricity of the specimen stage, inherent low dose per image, specimen charging, beam-induced specimen motions, and defocus gradients resulting both from specimen tilting and from unpredictable ice thickness variations. Current CTF correction methods for cryoET make at least one of the following assumptions: that the defocus at the center of the image is the same across the images of a tiltseries, that the particles all lie at the same Z-height in the embedding ice, and/or that the specimen, the cryo-electron microscopy (cryoEM) grid and/or the carbon support are flat. These experimental conditions are not always met. We have developed a CTF correction algorithm for cryoSPT without making any of the aforementioned assumptions. We also introduce speed and accuracy improvements and a higher degree of automation to the subtomogram averaging algorithms available in EMAN2. Using motion-corrected images of isolated virus particles as a benchmark specimen, recorded with a DE20 direct detection camera, we show that our CTF correction and subtomogram alignment routines can yield subtomogram averages close to 4/5 Nyquist frequency of the detector under our experimental conditions.

    View details for DOI 10.1016/j.jsb.2016.03.018

    View details for PubMedID 27016284

    View details for PubMedCentralID PMC4846534

  • Chaperonin TRiC/CCT Recognizes Fusion Oncoprotein AML1-ETO through Subunit-Specific Interactions. Biophysical journal Roh, S. H., Kasembeli, M. M., Galaz-Montoya, J. G., Chiu, W., Tweardy, D. J. 2016; 110 (11): 2377–85

    Abstract

    AML1-ETO is the translational product of a chimeric gene created by the stable chromosome translocation t (8;21)(q22;q22). It causes acute myeloid leukemia (AML) by dysregulating the expression of genes critical for myeloid cell development and differentiation and recently has been reported to bind multiple subunits of the mammalian cytosolic chaperonin TRiC (or CCT), primarily through its DNA binding domain (AML1-175). Through these interactions, TRiC plays an important role in the synthesis, folding, and activity of AML1-ETO. Using single-particle cryo-electron microscopy, we demonstrate here that a folding intermediate of AML1-ETO's DNA-binding domain (AML1-175) forms a stable complex with apo-TRiC. Our structure reveals that AML1-175 associates directly with a specific subset of TRiC subunits in the open conformation.

    View details for DOI 10.1016/j.bpj.2016.04.045

    View details for PubMedID 27276256

    View details for PubMedCentralID PMC4906440

  • Visualizing red blood cell sickling and the effects of inhibition of sphingosine kinase 1 using soft X-ray tomography. Journal of cell science Darrow, M. C., Zhang, Y., Cinquin, B. P., Smith, E. A., Boudreau, R., Rochat, R. H., Schmid, M. F., Xia, Y., Larabell, C. A., Chiu, W. 2016; 129 (18): 3511–17

    Abstract

    Sickle cell disease is a destructive genetic disorder characterized by the formation of fibrils of deoxygenated hemoglobin, leading to the red blood cell (RBC) morphology changes that underlie the clinical manifestations of this disease. Using cryogenic soft X-ray tomography (SXT), we characterized the morphology of sickled RBCs in terms of volume and the number of protrusions per cell. We were able to identify statistically a relationship between the number of protrusions and the volume of the cell, which is known to correlate to the severity of sickling. This structural polymorphism allows for the classification of the stages of the sickling process. Recent studies have shown that elevated sphingosine kinase 1 (Sphk1)-mediated sphingosine 1-phosphate production contributes to sickling. Here, we further demonstrate that compound 5C, an inhibitor of Sphk1, has anti-sickling properties. Additionally, the variation in cellular morphology upon treatment suggests that this drug acts to delay the sickling process. SXT is an effective tool that can be used to identify the morphology of the sickling process and assess the effectiveness of potential therapeutics.

    View details for DOI 10.1242/jcs.189225

    View details for PubMedID 27505892

    View details for PubMedCentralID PMC5047677

  • Improved Peak Detection and Deconvolution of Native Electrospray Mass Spectra from Large Protein Complexes JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY Lu, J., Trnka, M. J., Roh, S., Robinson, P. J., Shiau, C., Fujimori, D. G., Chiu, W., Burlingame, A. L., Guan, S. 2015; 26 (12): 2141-2151

    Abstract

    Native electrospray-ionization mass spectrometry (native MS) measures biomolecules under conditions that preserve most aspects of protein tertiary and quaternary structure, enabling direct characterization of large intact protein assemblies. However, native spectra derived from these assemblies are often partially obscured by low signal-to-noise as well as broad peak shapes because of residual solvation and adduction after the electrospray process. The wide peak widths together with the fact that sequential charge state series from highly charged ions are closely spaced means that native spectra containing multiple species often suffer from high degrees of peak overlap or else contain highly interleaved charge envelopes. This situation presents a challenge for peak detection, correct charge state and charge envelope assignment, and ultimately extraction of the relevant underlying mass values of the noncovalent assemblages being investigated. In this report, we describe a comprehensive algorithm developed for addressing peak detection, peak overlap, and charge state assignment in native mass spectra, called PeakSeeker. Overlapped peaks are detected by examination of the second derivative of the raw mass spectrum. Charge state distributions of the molecular species are determined by fitting linear combinations of charge envelopes to the overall experimental mass spectrum. This software is capable of deconvoluting heterogeneous, complex, and noisy native mass spectra of large protein assemblies as demonstrated by analysis of (1) synthetic mononucleosomes containing severely overlapping peaks, (2) an RNA polymerase II/α-amanitin complex with many closely interleaved ion signals, and (3) human TriC complex containing high levels of background noise. Graphical Abstract ᅟ.

    View details for DOI 10.1007/s13361-015-1235-6

    View details for Web of Science ID 000365116500020

    View details for PubMedID 26323614

  • Outcome of the First wwPDB Hybrid/Integrative Methods Task Force Workshop STRUCTURE Sali, A., Berman, H. M., Schwede, T., Trewhella, J., Kleywegt, G., Burley, S. K., Markley, J., Nakamura, H., Adams, P., Bonvin, A. M., Chiu, W., Dal Peraro, M., Di Maio, F., Ferrin, T. E., Gruenewald, K., Gutmanas, A., Henderson, R., Hummer, G., Iwasaki, K., Johnson, G., Lawson, C. L., Meiler, J., Marti-Renom, M. A., Montelione, G. T., Nilges, M., Nussinov, R., Patwardhan, A., Rappsilber, J., Read, R. J., Saibil, H., Schroeder, G. F., Schwieters, C. D., Seidel, C. A., Svergun, D., Topf, M., Ulrich, E. L., Velankar, S., Westbrook, J. D. 2015; 23 (7): 1156-1167

    Abstract

    Structures of biomolecular systems are increasingly computed by integrative modeling that relies on varied types of experimental data and theoretical information. We describe here the proceedings and conclusions from the first wwPDB Hybrid/Integrative Methods Task Force Workshop held at the European Bioinformatics Institute in Hinxton, UK, on October 6 and 7, 2014. At the workshop, experts in various experimental fields of structural biology, experts in integrative modeling and visualization, and experts in data archiving addressed a series of questions central to the future of structural biology. How should integrative models be represented? How should the data and integrative models be validated? What data should be archived? How should the data and models be archived? What information should accompany the publication of integrative models?

    View details for DOI 10.1016/j.str.2015.05.013

    View details for Web of Science ID 000360312200004

    View details for PubMedID 26095030

    View details for PubMedCentralID PMC4933300

  • CTF Challenge: Result summary JOURNAL OF STRUCTURAL BIOLOGY Marabini, R., Carragher, B., Chen, S., Chen, J., Cheng, A., Downing, K. H., Frank, J., Grassucci, R. A., Heymann, J. B., Jiang, W., Jonic, S., Liao, H. Y., Ludtke, S. J., Patwari, S., Piotrowski, A. L., Quintana, A., Sorzano, C. O., Stahlberg, H., Vargas, J., Voss, N. R., Chiu, W., Carazo, J. M. 2015; 190 (3): 348-359

    Abstract

    Image formation in bright field electron microscopy can be described with the help of the contrast transfer function (CTF). In this work the authors describe the "CTF Estimation Challenge", called by the Madrid Instruct Image Processing Center (I2PC) in collaboration with the National Center for Macromolecular Imaging (NCMI) at Houston. Correcting for the effects of the CTF requires accurate knowledge of the CTF parameters, but these have often been difficult to determine. In this challenge, researchers have had the opportunity to test their ability in estimating some of the key parameters of the electron microscope CTF on a large micrograph data set produced by well-known laboratories on a wide set of experimental conditions. This work presents the first analysis of the results of the CTF Estimation Challenge, including an assessment of the performance of the different software packages under different conditions, so as to identify those areas of research where further developments would be desirable in order to achieve high-resolution structural information.

    View details for DOI 10.1016/j.jsb.2015.04.003

    View details for Web of Science ID 000356115600009

    View details for PubMedID 25913484

  • Electron cryotomography reveals ultrastructure alterations in platelets from patients with ovarian cancer. Proceedings of the National Academy of Sciences of the United States of America Wang, R., Stone, R. L., Kaelber, J. T., Rochat, R. H., Nick, A. M., Vijayan, K. V., Afshar-Kharghan, V., Schmid, M. F., Dong, J. F., Sood, A. K., Chiu, W. 2015; 112 (46): 14266–71

    Abstract

    Thrombocytosis and platelet hyperreactivity are known to be associated with malignancy; however, there have been no ultrastructure studies of platelets from patients with ovarian cancer. Here, we used electron cryotomography (cryo-ET) to examine frozen-hydrated platelets from patients with invasive ovarian cancer (n = 12) and control subjects either with benign adnexal mass (n = 5) or free from disease (n = 6). Qualitative inspections of the tomograms indicate significant morphological differences between the cancer and control platelets, including disruption of the microtubule marginal band. Quantitative analysis of subcellular features in 120 platelet electron tomograms from these two groups showed statistically significant differences in mitochondria, as well as microtubules. These structural variations in the platelets from the patients with cancer may be correlated with the altered platelet functions associated with malignancy. Cryo-ET of platelets shows potential as a noninvasive biomarker technology for ovarian cancer and other platelet-related diseases.

    View details for DOI 10.1073/pnas.1518628112

    View details for PubMedID 26578771

    View details for PubMedCentralID PMC4655568

  • Lemon-shaped halo archaeal virus His1 with uniform tail but variable capsid structure. Proceedings of the National Academy of Sciences of the United States of America Hong, C., Pietilä, M. K., Fu, C. J., Schmid, M. F., Bamford, D. H., Chiu, W. 2015; 112 (8): 2449–54

    Abstract

    Lemon-shaped viruses are common in nature but so far have been observed to infect only archaea. Due to their unusual shape, the structures of these viruses are challenging to study and therefore poorly characterized. Here, we have studied haloarchaeal virus His1 using cryo-electron tomography as well as biochemical dissociation. The virions have different sizes, but prove to be extremely stable under various biochemical treatments. Subtomogram averaging of the computationally extracted virions resolved a tail-like structure with a central tail hub density and six tail spikes. Inside the tail there are two cavities and a plug density that separates the tail hub from the interior genome. His1 most likely uses the tail spikes to anchor to host cells and the tail hub to eject the genome, analogous to classic tailed bacteriophages. Upon biochemical treatment that releases the genome, the lemon-shaped virion transforms into an empty tube. Such a dramatic transformation demonstrates that the capsid proteins are capable of undergoing substantial quaternary structural changes, which may occur at different stages of the virus life cycle.

    View details for DOI 10.1073/pnas.1425008112

    View details for PubMedID 25675521

    View details for PubMedCentralID PMC4345568

  • A newly isolated reovirus has the simplest genomic and structural organization of any reovirus. Journal of virology Auguste, A. J., Kaelber, J. T., Fokam, E. B., Guzman, H., Carrington, C. V., Erasmus, J. H., Kamgang, B., Popov, V. L., Jakana, J., Liu, X., Wood, T. G., Widen, S. G., Vasilakis, N., Tesh, R. B., Chiu, W., Weaver, S. C. 2015; 89 (1): 676–87

    Abstract

    A total of 2,691 mosquitoes representing 17 species was collected from eight locations in southwest Cameroon and screened for pathogenic viruses. Ten isolates of a novel reovirus (genus Dinovernavirus) were detected by culturing mosquito pools on Aedes albopictus (C6/36) cell cultures. A virus that caused overt cytopathic effects was isolated, but it did not infect vertebrate cells or produce detectable disease in infant mice after intracerebral inoculation. The virus, tentatively designated Fako virus (FAKV), represents the first 9-segment, double-stranded RNA (dsRNA) virus to be isolated in nature. FAKV appears to have a broad mosquito host range, and its detection in male specimens suggests mosquito-to-mosquito transmission in nature. The structure of the T=1 FAKV virion, determined to subnanometer resolution by cryoelectron microscopy (cryo-EM), showed only four proteins per icosahedral asymmetric unit: a dimer of the major capsid protein, one turret protein, and one clamp protein. While all other turreted reoviruses of known structures have at least two copies of the clamp protein per asymmetric unit, FAKV's clamp protein bound at only one conformer of the major capsid protein. The FAKV capsid architecture and genome organization represent the most simplified reovirus described to date, and phylogenetic analysis suggests that it arose from a more complex ancestor by serial loss-of-function events.We describe the detection, genetic, phenotypic, and structural characteristics of a novel Dinovernavirus species isolated from mosquitoes collected in Cameroon. The virus, tentatively designated Fako virus (FAKV), is related to both single-shelled and partially double-shelled viruses. The only other described virus in this genus was isolated from cultured mosquito cells. It was previously unclear whether the phenotypic characteristics of that virus were reflective of this genus in nature or were altered during serial passaging in the chronically infected cell line. FAKV is a naturally occurring single-shelled reovirus with a unique virion architecture that lacks several key structural elements thought to stabilize a single-shelled reovirus virion, suggesting what may be the minimal number of proteins needed to form a viable reovirus particle. FAKV evolved from more complex ancestors by losing a genome segment and several virion proteins.

    View details for DOI 10.1128/JVI.02264-14

    View details for PubMedID 25355879

    View details for PubMedCentralID PMC4301156

  • Structure of a biologically active estrogen receptor-coactivator complex on DNA. Molecular cell Yi, P., Wang, Z., Feng, Q., Pintilie, G. D., Foulds, C. E., Lanz, R. B., Ludtke, S. J., Schmid, M. F., Chiu, W., O'Malley, B. W. 2015; 57 (6): 1047–58

    Abstract

    Estrogen receptor (ER/ESR1) is a transcription factor critical for development, reproduction, metabolism, and cancer. ER function hinges on its ability to recruit primary and secondary coactivators, yet structural information on the full-length receptor-coactivator complex to complement preexisting and sometimes controversial biochemical information is lacking. Here, we use cryoelectron microscopy (cryo-EM) to determine the quaternary structure of an active complex of DNA-bound ERα, steroid receptor coactivator 3 (SRC-3/NCOA3), and a secondary coactivator (p300/EP300). Our structural model suggests the following assembly mechanism for the complex: each of the two ligand-bound ERα monomers independently recruits one SRC-3 protein via the transactivation domain of ERα; the two SRC-3s in turn bind to different regions of one p300 protein through multiple contacts. We also present structural evidence for the location of activation function 1 (AF-1) in a full-length nuclear receptor, which supports a role for AF-1 in SRC-3 recruitment.

    View details for DOI 10.1016/j.molcel.2015.01.025

    View details for PubMedID 25728767

    View details for PubMedCentralID PMC4369429

  • An Intrinsically Disordered Peptide from Ebola Virus VP35 Controls Viral RNA Synthesis by Modulating Nucleoprotein-RNA Interactions. Cell reports Leung, D. W., Borek, D., Luthra, P., Binning, J. M., Anantpadma, M., Liu, G., Harvey, I. B., Su, Z., Endlich-Frazier, A., Pan, J., Shabman, R. S., Chiu, W., Davey, R. A., Otwinowski, Z., Basler, C. F., Amarasinghe, G. K. 2015; 11 (3): 376–89

    Abstract

    During viral RNA synthesis, Ebola virus (EBOV) nucleoprotein (NP) alternates between an RNA-template-bound form and a template-free form to provide the viral polymerase access to the RNA template. In addition, newly synthesized NP must be prevented from indiscriminately binding to noncognate RNAs. Here, we investigate the molecular bases for these critical processes. We identify an intrinsically disordered peptide derived from EBOV VP35 (NPBP, residues 20-48) that binds NP with high affinity and specificity, inhibits NP oligomerization, and releases RNA from NP-RNA complexes in vitro. The structure of the NPBP/ΔNPNTD complex, solved to 3.7 Å resolution, reveals how NPBP peptide occludes a large surface area that is important for NP-NP and NP-RNA interactions and for viral RNA synthesis. Together, our results identify a highly conserved viral interface that is important for EBOV replication and can be targeted for therapeutic development.

    View details for DOI 10.1016/j.celrep.2015.03.034

    View details for PubMedID 25865894

    View details for PubMedCentralID PMC4599368

  • Structural Mechanisms of Mutant Huntingtin Aggregation Suppression by the Synthetic Chaperonin-like CCT5 Complex Explained by Cryoelectron Tomography. The Journal of biological chemistry Darrow, M. C., Sergeeva, O. A., Isas, J. M., Galaz-Montoya, J. G., King, J. A., Langen, R., Schmid, M. F., Chiu, W. 2015; 290 (28): 17451–61

    Abstract

    Huntington disease, a neurodegenerative disorder characterized by functional deficits and loss of striatal neurons, is linked to an expanded and unstable CAG trinucleotide repeat in the huntingtin gene (HTT). This DNA sequence translates to a polyglutamine repeat in the protein product, leading to mutant huntingtin (mHTT) protein aggregation. The aggregation of mHTT is inhibited in vitro and in vivo by the TCP-1 ring complex (TRiC) chaperonin. Recently, a novel complex comprised of a single type of TRiC subunit has been reported to inhibit mHTT aggregation. Specifically, the purified CCT5 homo-oligomer complex, when compared with TRiC, has a similar structure, ATP use, and substrate refolding activity, and, importantly, it also inhibits mHTT aggregation. Using an aggregation suppression assay and cryoelectron tomography coupled with a novel computational classification method, we uncover the interactions between the synthetic CCT5 complex (∼ 1 MDa) and aggregates of mutant huntingtin exon 1 containing 46 glutamines (mHTTQ46-Ex1). We find that, in a similar fashion to TRiC, synthetic CCT5 complex caps mHTT fibrils at their tips and encapsulates mHTT oligomers, providing a structural description of the inhibition of mHTTQ46-Ex1 by CCT5 complex and a shared mechanism of mHTT inhibition between TRiC chaperonin and the CCT5 complex: cap and contain.

    View details for DOI 10.1074/jbc.M115.655373

    View details for PubMedID 25995452

    View details for PubMedCentralID PMC4498080

  • Structural diversity of supercoiled DNA. Nature communications Irobalieva, R. N., Fogg, J. M., Catanese, D. J., Sutthibutpong, T., Chen, M., Barker, A. K., Ludtke, S. J., Harris, S. A., Schmid, M. F., Chiu, W., Zechiedrich, L. 2015; 6: 8440

    Abstract

    By regulating access to the genetic code, DNA supercoiling strongly affects DNA metabolism. Despite its importance, however, much about supercoiled DNA (positively supercoiled DNA, in particular) remains unknown. Here we use electron cryo-tomography together with biochemical analyses to investigate structures of individual purified DNA minicircle topoisomers with defined degrees of supercoiling. Our results reveal that each topoisomer, negative or positive, adopts a unique and surprisingly wide distribution of three-dimensional conformations. Moreover, we uncover striking differences in how the topoisomers handle torsional stress. As negative supercoiling increases, bases are increasingly exposed. Beyond a sharp supercoiling threshold, we also detect exposed bases in positively supercoiled DNA. Molecular dynamics simulations independently confirm the conformational heterogeneity and provide atomistic insight into the flexibility of supercoiled DNA. Our integrated approach reveals the three-dimensional structures of DNA that are essential for its function.

    View details for DOI 10.1038/ncomms9440

    View details for PubMedID 26455586

    View details for PubMedCentralID PMC4608029

  • Contribution of the Type II Chaperonin, TRiC/CCT, to Oncogenesis. International journal of molecular sciences Roh, S. H., Kasembeli, M., Bakthavatsalam, D., Chiu, W., Tweardy, D. J. 2015; 16 (11): 26706–20

    Abstract

    The folding of newly synthesized proteins and the maintenance of pre-existing proteins are essential in sustaining a living cell. A network of molecular chaperones tightly guides the folding, intracellular localization, and proteolytic turnover of proteins. Many of the key regulators of cell growth and differentiation have been identified as clients of molecular chaperones, which implies that chaperones are potential mediators of oncogenesis. In this review, we briefly provide an overview of the role of chaperones, including HSP70 and HSP90, in cancer. We further summarize and highlight the emerging the role of chaperonin TRiC (T-complex protein-1 ring complex, also known as CCT) in the development and progression of cancer mediated through its critical interactions with oncogenic clients that modulate growth deregulation, apoptosis, and genome instability in cancer cells. Elucidation of how TRiC modulates the folding and function of oncogenic clients will provide strategies for developing novel cancer therapies.

    View details for DOI 10.3390/ijms161125975

    View details for PubMedID 26561808

    View details for PubMedCentralID PMC4661834

  • An atomic model of brome mosaic virus using direct electron detection and real-space optimization NATURE COMMUNICATIONS Wang, Z., Hryc, C. F., Bammes, B., Afonine, P. V., Jakana, J., Chen, D., Liu, X., Baker, M. L., Kao, C., Ludtke, S. J., Schmid, M. F., Adams, P. D., Chiu, W. 2014; 5

    Abstract

    Advances in electron cryo-microscopy have enabled structure determination of macromolecules at near-atomic resolution. However, structure determination, even using de novo methods, remains susceptible to model bias and overfitting. Here we describe a complete workflow for data acquisition, image processing, all-atom modelling and validation of brome mosaic virus, an RNA virus. Data were collected with a direct electron detector in integrating mode and an exposure beyond the traditional radiation damage limit. The final density map has a resolution of 3.8 Å as assessed by two independent data sets and maps. We used the map to derive an all-atom model with a newly implemented real-space optimization protocol. The validity of the model was verified by its match with the density map and a previous model from X-ray crystallography, as well as the internal consistency of models from independent maps. This study demonstrates a practical approach to obtain a rigorously validated atomic resolution electron cryo-microscopy structure.

    View details for DOI 10.1038/ncomms5808

    View details for Web of Science ID 000342928700012

    View details for PubMedID 25185801

  • Modulation of STAT3 folding and function by TRiC/CCT chaperonin. PLoS biology Kasembeli, M., Lau, W. C., Roh, S., Eckols, T. K., Frydman, J., Chiu, W., Tweardy, D. J. 2014; 12 (4)

    Abstract

    Signal transducer and activator of transcription 3 (Stat3) transduces signals of many peptide hormones from the cell surface to the nucleus and functions as an oncoprotein in many types of cancers, yet little is known about how it achieves its native folded state within the cell. Here we show that Stat3 is a novel substrate of the ring-shaped hetero-oligomeric eukaryotic chaperonin, TRiC/CCT, which contributes to its biosynthesis and activity in vitro and in vivo. TRiC binding to Stat3 was mediated, at least in part, by TRiC subunit CCT3. Stat3 binding to TRiC mapped predominantly to the β-strand rich, DNA-binding domain of Stat3. Notably, enhancing Stat3 binding to TRiC by engineering an additional TRiC-binding domain from the von Hippel-Lindau protein (vTBD), at the N-terminus of Stat3, further increased its affinity for TRiC as well as its function, as determined by Stat3's ability to bind to its phosphotyrosyl-peptide ligand, an interaction critical for Stat3 activation. Thus, Stat3 levels and function are regulated by TRiC and can be modulated by manipulating its interaction with TRiC.

    View details for DOI 10.1371/journal.pbio.1001844

    View details for PubMedID 24756126

    View details for PubMedCentralID PMC3995649

  • A structural model of the genome packaging process in a membrane-containing double stranded DNA virus. PLoS biology Hong, C., Oksanen, H. M., Liu, X., Jakana, J., Bamford, D. H., Chiu, W. 2014; 12 (12): e1002024

    Abstract

    Two crucial steps in the virus life cycle are genome encapsidation to form an infective virion and genome exit to infect the next host cell. In most icosahedral double-stranded (ds) DNA viruses, the viral genome enters and exits the capsid through a unique vertex. Internal membrane-containing viruses possess additional complexity as the genome must be translocated through the viral membrane bilayer. Here, we report the structure of the genome packaging complex with a membrane conduit essential for viral genome encapsidation in the tailless icosahedral membrane-containing bacteriophage PRD1. We utilize single particle electron cryo-microscopy (cryo-EM) and symmetry-free image reconstruction to determine structures of PRD1 virion, procapsid, and packaging deficient mutant particles. At the unique vertex of PRD1, the packaging complex replaces the regular 5-fold structure and crosses the lipid bilayer. These structures reveal that the packaging ATPase P9 and the packaging efficiency factor P6 form a dodecameric portal complex external to the membrane moiety, surrounded by ten major capsid protein P3 trimers. The viral transmembrane density at the special vertex is assigned to be a hexamer of heterodimer of proteins P20 and P22. The hexamer functions as a membrane conduit for the DNA and as a nucleating site for the unique vertex assembly. Our structures show a conformational alteration in the lipid membrane after the P9 and P6 are recruited to the virion. The P8-genome complex is then packaged into the procapsid through the unique vertex while the genome terminal protein P8 functions as a valve that closes the channel once the genome is inside. Comparing mature virion, procapsid, and mutant particle structures led us to propose an assembly pathway for the genome packaging apparatus in the PRD1 virion.

    View details for DOI 10.1371/journal.pbio.1002024

    View details for PubMedID 25514469

    View details for PubMedCentralID PMC4267777

  • Preparation of primary neurons for visualizing neurites in a frozen-hydrated state using cryo-electron tomography. Journal of visualized experiments : JoVE Shahmoradian, S. H., Galiano, M. R., Wu, C., Chen, S., Rasband, M. N., Mobley, W. C., Chiu, W. 2014: e50783

    Abstract

    Neurites, both dendrites and axons, are neuronal cellular processes that enable the conduction of electrical impulses between neurons. Defining the structure of neurites is critical to understanding how these processes move materials and signals that support synaptic communication. Electron microscopy (EM) has been traditionally used to assess the ultrastructural features within neurites; however, the exposure to organic solvent during dehydration and resin embedding can distort structures. An important unmet goal is the formulation of procedures that allow for structural evaluations not impacted by such artifacts. Here, we have established a detailed and reproducible protocol for growing and flash-freezing whole neurites of different primary neurons on electron microscopy grids followed by their examination with cryo-electron tomography (cryo-ET). This technique allows for 3-D visualization of frozen, hydrated neurites at nanometer resolution, facilitating assessment of their morphological differences. Our protocol yields an unprecedented view of dorsal root ganglion (DRG) neurites, and a visualization of hippocampal neurites in their near-native state. As such, these methods create a foundation for future studies on neurites of both normal neurons and those impacted by neurological disorders.

    View details for DOI 10.3791/50783

    View details for PubMedID 24561719

    View details for PubMedCentralID PMC4089403

  • Editorial overview: virus structure and function. Current opinion in virology Chiu, W., Crépin, T., Ruigrok, R. W. 2014; 5: viii-ix

    View details for DOI 10.1016/j.coviro.2014.03.003

    View details for PubMedID 24726158

    View details for PubMedCentralID PMC4209247

  • Crystal structure of a nematode-infecting virus. Proceedings of the National Academy of Sciences of the United States of America Guo, Y. R., Hryc, C. F., Jakana, J., Jiang, H., Wang, D., Chiu, W., Zhong, W., Tao, Y. J. 2014; 111 (35): 12781–86

    Abstract

    Orsay, the first virus discovered to naturally infect Caenorhabditis elegans or any nematode, has a bipartite, positive-sense RNA genome. Sequence analyses show that Orsay is related to nodaviruses, but molecular characterizations of Orsay reveal several unique features, such as the expression of a capsid-δ fusion protein and the use of an ATG-independent mechanism for translation initiation. Here we report the crystal structure of an Orsay virus-like particle assembled from recombinant capsid protein (CP). Orsay capsid has a T = 3 icosahedral symmetry with 60 trimeric surface spikes. Each CP can be divided into three regions: an N-terminal arm that forms an extended protein interaction network at the capsid interior, an S domain with a jelly-roll, β-barrel fold forming the continuous capsid, and a P domain that forms surface spike projections. The structure of the Orsay S domain is best aligned to T = 3 plant RNA viruses but exhibits substantial differences compared with the insect-infecting alphanodaviruses, which also lack the P domain in their CPs. The Orsay P domain is remotely related to the P1 domain in calicivirus and hepatitis E virus, suggesting a possible evolutionary relationship. Removing the N-terminal arm produced a slightly expanded capsid with fewer nucleic acids packaged, suggesting that the arm is important for capsid stability and genome packaging. Because C. elegans-Orsay serves as a highly tractable model for studying viral pathogenesis, our results should provide a valuable structural framework for further studies of Orsay replication and infection.

    View details for DOI 10.1073/pnas.1407122111

    View details for PubMedID 25136116

    View details for PubMedCentralID PMC4156749

  • A 3D cellular context for the macromolecular world. Nature structural & molecular biology Patwardhan, A., Ashton, A., Brandt, R., Butcher, S., Carzaniga, R., Chiu, W., Collinson, L., Doux, P., Duke, E., Ellisman, M. H., Franken, E., Grünewald, K., Heriche, J. K., Koster, A., Kühlbrandt, W., Lagerstedt, I., Larabell, C., Lawson, C. L., Saibil, H. R., Sanz-García, E., Subramaniam, S., Verkade, P., Swedlow, J. R., Kleywegt, G. J. 2014; 21 (10): 841–45

    View details for DOI 10.1038/nsmb.2897

    View details for PubMedID 25289590

    View details for PubMedCentralID PMC4346196

  • Cryo-EM techniques to resolve the structure of HSV-1 capsid-associated components. Methods in molecular biology (Clifton, N.J.) Rochat, R. H., Hecksel, C. W., Chiu, W. 2014; 1144: 265–81

    Abstract

    Electron cryo-microscopy has become a routine technique to determine the structure of biochemically purified herpes simplex virus capsid particles. This chapter describes the procedures of specimen preparation by cryopreservation; low dose and low temperature imaging in an electron cryo-microscope; and data processing for reconstruction. This methodology has yielded subnanometer resolution structures of the icosahedral capsid shell where α-helices and β-sheets of individual subunits can be recognized. A relaxation of the symmetry in the reconstruction steps allows us to resolve the DNA packaging protein located at one of the 12 vertices in the capsid.

    View details for DOI 10.1007/978-1-4939-0428-0_18

    View details for PubMedID 24671690

    View details for PubMedCentralID PMC4370171

  • Structure of the AcrAB-TolC multidrug efflux pump. Nature Du, D., Wang, Z., James, N. R., Voss, J. E., Klimont, E., Ohene-Agyei, T., Venter, H., Chiu, W., Luisi, B. F. 2014; 509 (7501): 512–15

    Abstract

    The capacity of numerous bacterial species to tolerate antibiotics and other toxic compounds arises in part from the activity of energy-dependent transporters. In Gram-negative bacteria, many of these transporters form multicomponent 'pumps' that span both inner and outer membranes and are driven energetically by a primary or secondary transporter component. A model system for such a pump is the acridine resistance complex of Escherichia coli. This pump assembly comprises the outer-membrane channel TolC, the secondary transporter AcrB located in the inner membrane, and the periplasmic AcrA, which bridges these two integral membrane proteins. The AcrAB-TolC efflux pump is able to transport vectorially a diverse array of compounds with little chemical similarity, thus conferring resistance to a broad spectrum of antibiotics. Homologous complexes are found in many Gram-negative species, including in animal and plant pathogens. Crystal structures are available for the individual components of the pump and have provided insights into substrate recognition, energy coupling and the transduction of conformational changes associated with the transport process. However, how the subunits are organized in the pump, their stoichiometry and the details of their interactions are not known. Here we present the pseudo-atomic structure of a complete multidrug efflux pump in complex with a modulatory protein partner from E. coli. The model defines the quaternary organization of the pump, identifies key domain interactions, and suggests a cooperative process for channel assembly and opening. These findings illuminate the basis for drug resistance in numerous pathogenic bacterial species.

    View details for DOI 10.1038/nature13205

    View details for PubMedID 24747401

    View details for PubMedCentralID PMC4361902

  • Multiple functional roles of the accessory I-domain of bacteriophage P22 coat protein revealed by NMR structure and CryoEM modeling. Structure (London, England : 1993) Rizzo, A. A., Suhanovsky, M. M., Baker, M. L., Fraser, L. C., Jones, L. M., Rempel, D. L., Gross, M. L., Chiu, W., Alexandrescu, A. T., Teschke, C. M. 2014; 22 (6): 830–41

    Abstract

    Some capsid proteins built on the ubiquitous HK97-fold have accessory domains imparting specific functions. Bacteriophage P22 coat protein has a unique insertion domain (I-domain). Two prior I-domain models from subnanometer cryoelectron microscopy (cryoEM) reconstructions differed substantially. Therefore, the I-domain's nuclear magnetic resonance structure was determined and also used to improve cryoEM models of coat protein. The I-domain has an antiparallel six-stranded β-barrel fold, not previously observed in HK97-fold accessory domains. The D-loop, which is dynamic in the isolated I-domain and intact monomeric coat protein, forms stabilizing salt bridges between adjacent capsomers in procapsids. The S-loop is important for capsid size determination, likely through intrasubunit interactions. Ten of 18 coat protein temperature-sensitive-folding substitutions are in the I-domain, indicating its importance in folding and stability. Several are found on a positively charged face of the β-barrel that anchors the I-domain to a negatively charged surface of the coat protein HK97-core.

    View details for DOI 10.1016/j.str.2014.04.003

    View details for PubMedID 24836025

    View details for PubMedCentralID PMC4068711

  • Chaperonin-containing TCP-1 complex directly binds to the cytoplasmic domain of the LOX-1 receptor. FEBS letters Bakthavatsalam, D., Soung, R. H., Tweardy, D. J., Chiu, W., Dixon, R. A., Woodside, D. G. 2014; 588 (13): 2133–40

    Abstract

    Lectin-like oxidized low-density lipoprotein receptor (LOX-1) is a scavenger receptor that binds oxidized low-density lipoprotein (OxLDL) and has a role in atherosclerosis development. The N-terminus intracellular region (cytoplasmic domain) of LOX-1 mediates receptor internalization and trafficking, potentially through intracellular protein interactions. Using affinity isolation, we identified 6 of the 8 components of the chaperonin-containing TCP-1 (CCT) complex bound to LOX-1 cytoplasmic domain, which we verified by coimmunoprecipitation and immunostaining in human umbilical vein endothelial cells. We found that the interaction between CCT and LOX-1 is direct and ATP-dependent and that OxLDL suppressed this interaction. Understanding the association between LOX-1 and the CCT complex may facilitate the design of novel therapies for cardiovascular disease.

    View details for DOI 10.1016/j.febslet.2014.04.049

    View details for PubMedID 24846140

    View details for PubMedCentralID PMC4100626

  • Protruding knob-like proteins violate local symmetries in an icosahedral marine virus. Nature communications Gipson, P., Baker, M. L., Raytcheva, D., Haase-Pettingell, C., Piret, J., King, J. A., Chiu, W. 2014; 5: 4278

    Abstract

    Marine viruses play crucial roles in shaping the dynamics of oceanic microbial communities and in the carbon cycle on Earth. Here we report a 4.7-Å structure of a cyanobacterial virus, Syn5, by electron cryo-microscopy and modelling. A Cα backbone trace of the major capsid protein (gp39) reveals a classic phage protein fold. In addition, two knob-like proteins protruding from the capsid surface are also observed. Using bioinformatics and structure analysis tools, these proteins are identified to correspond to gp55 and gp58 (each with two copies per asymmetric unit). The non 1:1 stoichiometric distribution of gp55/58 to gp39 breaks all expected local symmetries and leads to non-quasi-equivalence of the capsid subunits, suggesting a role in capsid stabilization. Such a structural arrangement has not yet been observed in any known virus structures.

    View details for DOI 10.1038/ncomms5278

    View details for PubMedID 24985522

    View details for PubMedCentralID PMC4102127

  • Capsid expansion mechanism of bacteriophage T7 revealed by multistate atomic models derived from cryo-EM reconstructions. Proceedings of the National Academy of Sciences of the United States of America Guo, F., Liu, Z., Fang, P. A., Zhang, Q., Wright, E. T., Wu, W., Zhang, C., Vago, F., Ren, Y., Jakana, J., Chiu, W., Serwer, P., Jiang, W. 2014; 111 (43): E4606–14

    Abstract

    Many dsDNA viruses first assemble a DNA-free procapsid, using a scaffolding protein-dependent process. The procapsid, then, undergoes dramatic conformational maturation while packaging DNA. For bacteriophage T7 we report the following four single-particle cryo-EM 3D reconstructions and the derived atomic models: procapsid (4.6-Å resolution), an early-stage DNA packaging intermediate (3.5 Å), a later-stage packaging intermediate (6.6 Å), and the final infectious phage (3.6 Å). In the procapsid, the N terminus of the major capsid protein, gp10, has a six-turn helix at the inner surface of the shell, where each skewed hexamer of gp10 interacts with two scaffolding proteins. With the exit of scaffolding proteins during maturation the gp10 N-terminal helix unfolds and swings through the capsid shell to the outer surface. The refolded N-terminal region has a hairpin that forms a novel noncovalent, joint-like, intercapsomeric interaction with a pocket formed during shell expansion. These large conformational changes also result in a new noncovalent, intracapsomeric topological linking. Both interactions further stabilize the capsids by interlocking all pentameric and hexameric capsomeres in both DNA packaging intermediate and phage. Although the final phage shell has nearly identical structure to the shell of the DNA-free intermediate, surprisingly we found that the icosahedral faces of the phage are slightly (∼4 Å) contracted relative to the faces of the intermediate, despite the internal pressure from the densely packaged DNA genome. These structures provide a basis for understanding the capsid maturation process during DNA packaging that is essential for large numbers of dsDNA viruses.

    View details for DOI 10.1073/pnas.1407020111

    View details for PubMedID 25313071

    View details for PubMedCentralID PMC4217468

  • Zernike phase-contrast electron cryotomography applied to marine cyanobacteria infected with cyanophages. Nature protocols Dai, W., Fu, C., Khant, H. A., Ludtke, S. J., Schmid, M. F., Chiu, W. 2014; 9 (11): 2630–42

    Abstract

    Advances in electron cryotomography have provided new opportunities to visualize the internal 3D structures of a bacterium. An electron microscope equipped with Zernike phase-contrast optics produces images with markedly increased contrast compared with images obtained by conventional electron microscopy. Here we describe a protocol to apply Zernike phase plate technology for acquiring electron tomographic tilt series of cyanophage-infected cyanobacterial cells embedded in ice, without staining or chemical fixation. We detail the procedures for aligning and assessing phase plates for data collection, and methods for obtaining 3D structures of cyanophage assembly intermediates in the host by subtomogram alignment, classification and averaging. Acquiring three or four tomographic tilt series takes ∼12 h on a JEM2200FS electron microscope. We expect this time requirement to decrease substantially as the technique matures. The time required for annotation and subtomogram averaging varies widely depending on the project goals and data volume.

    View details for DOI 10.1038/nprot.2014.176

    View details for PubMedID 25321408

    View details for PubMedCentralID PMC4371552

  • Identifying the assembly pathway of cyanophage inside the marine bacterium using electron cryo-tomography. Microbial cell (Graz, Austria) Dai, W., Schmid, M. F., King, J. A., Chiu, W. 2014; 1 (1): 45–47

    Abstract

    Advances in electron cryo-tomography open up a new avenue to visualize the 3-D internal structure of a single bacterium before and after its infection by bacteriophages in its native environment, without using chemical fixatives, fluorescent dyes or negative stains. Such direct observation reveals the presence of assembly intermediates of the bacteriophage and thus allows us to map out the maturation pathway of the bacteriophage inside its host.

    View details for DOI 10.15698/mic2014.01.125

    View details for PubMedID 25419524

    View details for PubMedCentralID PMC4238041

  • Validated near-atomic resolution structure of bacteriophage epsilon15 derived from cryo-EM and modeling PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Baker, M. L., Hryc, C. F., Zhang, Q., Wu, W., Jakana, J., Haase-Pettingell, C., Afonine, P. V., Adams, P. D., King, J. A., Jiang, W., Chiu, W. 2013; 110 (30): 12301-12306

    Abstract

    High-resolution structures of viruses have made important contributions to modern structural biology. Bacteriophages, the most diverse and abundant organisms on earth, replicate and infect all bacteria and archaea, making them excellent potential alternatives to antibiotics and therapies for multidrug-resistant bacteria. Here, we improved upon our previous electron cryomicroscopy structure of Salmonella bacteriophage epsilon15, achieving a resolution sufficient to determine the tertiary structures of both gp7 and gp10 protein subunits that form the T = 7 icosahedral lattice. This study utilizes recently established best practice for near-atomic to high-resolution (3-5 Å) electron cryomicroscopy data evaluation. The resolution and reliability of the density map were cross-validated by multiple reconstructions from truly independent data sets, whereas the models of the individual protein subunits were validated adopting the best practices from X-ray crystallography. Some sidechain densities are clearly resolved and show the subunit-subunit interactions within and across the capsomeres that are required to stabilize the virus. The presence of the canonical phage and jellyroll viral protein folds, gp7 and gp10, respectively, in the same virus suggests that epsilon15 may have emerged more recently relative to other bacteriophages.

    View details for DOI 10.1073/pnas.1309947110

    View details for Web of Science ID 000322112300044

    View details for PubMedID 23840063

  • TRiC's tricks inhibit huntingtin aggregation ELIFE Shahmoradian, S. H., Galaz-Montoya, J. G., Schmid, M. F., Cong, Y., Ma, B., Spiess, C., Frydman, J., Ludtke, S. J., Chiu, W. 2013; 2

    Abstract

    In Huntington's disease, a mutated version of the huntingtin protein leads to cell death. Mutant huntingtin is known to aggregate, a process that can be inhibited by the eukaryotic chaperonin TRiC (TCP1-ring complex) in vitro and in vivo. A structural understanding of the genesis of aggregates and their modulation by cellular chaperones could facilitate the development of therapies but has been hindered by the heterogeneity of amyloid aggregates. Using cryo-electron microscopy (cryoEM) and single particle cryo-electron tomography (SPT) we characterize the growth of fibrillar aggregates of mutant huntingtin exon 1 containing an expanded polyglutamine tract with 51 residues (mhttQ51), and resolve 3-D structures of the chaperonin TRiC interacting with mhttQ51. We find that TRiC caps mhttQ51 fibril tips via the apical domains of its subunits, and also encapsulates smaller mhtt oligomers within its chamber. These two complementary mechanisms provide a structural description for TRiC's inhibition of mhttQ51 aggregation in vitro. DOI:http://dx.doi.org/10.7554/eLife.00710.001.

    View details for DOI 10.7554/eLife.00710

    View details for Web of Science ID 000328620500004

    View details for PubMedID 23853712

    View details for PubMedCentralID PMC3707056

  • Cryo-EM model validation using independent map reconstructions. Protein science DiMaio, F., Zhang, J., Chiu, W., Baker, D. 2013; 22 (6): 865-868

    Abstract

    An increasing number of cryo-electron microscopy (cryo-EM) density maps are being generated with suitable resolution to trace the protein backbone and guide sidechain placement. Generating and evaluating atomic models based on such maps would be greatly facilitated by independent validation metrics for assessing the fit of the models to the data. We describe such a metric based on the fit of atomic models with independent test maps from single particle reconstructions not used in model refinement. The metric provides a means to determine the proper balance between the fit to the density and model energy and stereochemistry during refinement, and is likely to be useful in determining values of model building and refinement metaparameters quite generally.

    View details for DOI 10.1002/pro.2267

    View details for PubMedID 23592445

  • TRiC's tricks inhibit huntingtin aggregation. eLife Shahmoradian, S. H., Galaz-Montoya, J. G., Schmid, M. F., Cong, Y., Ma, B., Spiess, C., Frydman, J., Ludtke, S. J., Chiu, W. 2013; 2

    Abstract

    In Huntington's disease, a mutated version of the huntingtin protein leads to cell death. Mutant huntingtin is known to aggregate, a process that can be inhibited by the eukaryotic chaperonin TRiC (TCP1-ring complex) in vitro and in vivo. A structural understanding of the genesis of aggregates and their modulation by cellular chaperones could facilitate the development of therapies but has been hindered by the heterogeneity of amyloid aggregates. Using cryo-electron microscopy (cryoEM) and single particle cryo-electron tomography (SPT) we characterize the growth of fibrillar aggregates of mutant huntingtin exon 1 containing an expanded polyglutamine tract with 51 residues (mhttQ51), and resolve 3-D structures of the chaperonin TRiC interacting with mhttQ51. We find that TRiC caps mhttQ51 fibril tips via the apical domains of its subunits, and also encapsulates smaller mhtt oligomers within its chamber. These two complementary mechanisms provide a structural description for TRiC's inhibition of mhttQ51 aggregation in vitro. DOI:http://dx.doi.org/10.7554/eLife.00710.001.

    View details for DOI 10.7554/eLife.00710

    View details for PubMedID 23853712

    View details for PubMedCentralID PMC3707056

  • EMEN2: an object oriented database and electronic lab notebook. Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada Rees, I., Langley, E., Chiu, W., Ludtke, S. J. 2013; 19 (1): 1–10

    Abstract

    Transmission electron microscopy and associated methods, such as single particle analysis, two-dimensional crystallography, helical reconstruction, and tomography, are highly data-intensive experimental sciences, which also have substantial variability in experimental technique. Object-oriented databases present an attractive alternative to traditional relational databases for situations where the experiments themselves are continually evolving. We present EMEN2, an easy to use object-oriented database with a highly flexible infrastructure originally targeted for transmission electron microscopy and tomography, which has been extended to be adaptable for use in virtually any experimental science. It is a pure object-oriented database designed for easy adoption in diverse laboratory environments and does not require professional database administration. It includes a full featured, dynamic web interface in addition to APIs for programmatic access. EMEN2 installations currently support roughly 800 scientists worldwide with over 1/2 million experimental records and over 20 TB of experimental data. The software is freely available with complete source.

    View details for DOI 10.1017/S1431927612014043

    View details for PubMedID 23360752

    View details for PubMedCentralID PMC3907281

  • Validation of cryo-EM structure of IP₃R1 channel. Structure (London, England : 1993) Murray, S. C., Flanagan, J., Popova, O. B., Chiu, W., Ludtke, S. J., Serysheva, I. I. 2013; 21 (6): 900–909

    Abstract

    About a decade ago, three electron cryomicroscopy (cryo-EM) single-particle reconstructions of IP3R1 were reported at low resolution. It was disturbing that these structures bore little similarity to one another, even at the level of quaternary structure. Recently, we published an improved structure of IP3R1 at ∼1 nm resolution. However, this structure did not bear any resemblance to any of the three previously published structures, leading to the question of why the structure should be considered more reliable than the original three. Here, we apply several methods, including class-average/map comparisons, tilt-pair validation, and use of multiple refinement software packages, to give strong evidence for the reliability of our recent structure. The map resolution and feature resolvability are assessed with the gold standard criterion. This approach is generally applicable to assessing the validity of cryo-EM maps of other molecular machines.

    View details for DOI 10.1016/j.str.2013.04.016

    View details for PubMedID 23707684

    View details for PubMedCentralID PMC3696195

  • Visualizing GroEL/ES in the act of encapsulating a folding protein. Cell Chen, D. H., Madan, D., Weaver, J., Lin, Z., Schröder, G. F., Chiu, W., Rye, H. S. 2013; 153 (6): 1354–65

    Abstract

    The GroEL/ES chaperonin system is required for the assisted folding of many proteins. How these substrate proteins are encapsulated within the GroEL-GroES cavity is poorly understood. Using symmetry-free, single-particle cryo-electron microscopy, we have characterized a chemically modified mutant of GroEL (EL43Py) that is trapped at a normally transient stage of substrate protein encapsulation. We show that the symmetric pattern of the GroEL subunits is broken as the GroEL cis-ring apical domains reorient to accommodate the simultaneous binding of GroES and an incompletely folded substrate protein (RuBisCO). The collapsed RuBisCO folding intermediate binds to the lower segment of two apical domains, as well as to the normally unstructured GroEL C-terminal tails. A comparative structural analysis suggests that the allosteric transitions leading to substrate protein release and folding involve concerted shifts of GroES and the GroEL apical domains and C-terminal tails.

    View details for DOI 10.1016/j.cell.2013.04.052

    View details for PubMedID 23746846

    View details for PubMedCentralID PMC3695626

  • The Molecular Architecture of the Eukaryotic Chaperonin TRiC/CCT STRUCTURE Leitner, A., Joachimiak, L. A., Bracher, A., Moenkemeyer, L., Walzthoeni, T., Chen, B., Pechmann, S., Holmes, S., Cong, Y., Ma, B., Ludtke, S., Chiu, W., Hartl, F. U., Aebersold, R., Frydman, J. 2012; 20 (5): 814-825

    Abstract

    TRiC/CCT is a highly conserved and essential chaperonin that uses ATP cycling to facilitate folding of approximately 10% of the eukaryotic proteome. This 1 MDa hetero-oligomeric complex consists of two stacked rings of eight paralogous subunits each. Previously proposed TRiC models differ substantially in their subunit arrangements and ring register. Here, we integrate chemical crosslinking, mass spectrometry, and combinatorial modeling to reveal the definitive subunit arrangement of TRiC. In vivo disulfide mapping provided additional validation for the crosslinking-derived arrangement as the definitive TRiC topology. This subunit arrangement allowed the refinement of a structural model using existing X-ray diffraction data. The structure described here explains all available crosslink experiments, provides a rationale for previously unexplained structural features, and reveals a surprising asymmetry of charges within the chaperonin folding chamber.

    View details for DOI 10.1016/j.str.2012.03.007

    View details for Web of Science ID 000304214400008

    View details for PubMedID 22503819

    View details for PubMedCentralID PMC3350567

  • Symmetry-free cryo-EM structures of the chaperonin TRiC along its ATPase-driven conformational cycle EMBO JOURNAL Cong, Y., Schroeder, G. F., Meyer, A. S., Jakana, J., Ma, B., Dougherty, M. T., Schmid, M. F., Reissmann, S., Levitt, M., Ludtke, S. L., Frydman, J., Chiu, W. 2012; 31 (3): 720-730

    Abstract

    The eukaryotic group II chaperonin TRiC/CCT is a 16-subunit complex with eight distinct but similar subunits arranged in two stacked rings. Substrate folding inside the central chamber is triggered by ATP hydrolysis. We present five cryo-EM structures of TRiC in apo and nucleotide-induced states without imposing symmetry during the 3D reconstruction. These structures reveal the intra- and inter-ring subunit interaction pattern changes during the ATPase cycle. In the apo state, the subunit arrangement in each ring is highly asymmetric, whereas all nucleotide-containing states tend to be more symmetrical. We identify and structurally characterize an one-ring closed intermediate induced by ATP hydrolysis wherein the closed TRiC ring exhibits an observable chamber expansion. This likely represents the physiological substrate folding state. Our structural results suggest mechanisms for inter-ring-negative cooperativity, intra-ring-positive cooperativity, and protein-folding chamber closure of TRiC. Intriguingly, these mechanisms are different from other group I and II chaperonins despite their similar architecture.

    View details for DOI 10.1038/emboj.2011.366

    View details for Web of Science ID 000300871700019

    View details for PubMedID 22045336

    View details for PubMedCentralID PMC3273382

  • Three-dimensional architecture of the rod sensory cilium and its disruption in retinal neurodegeneration. Cell Gilliam, J. C., Chang, J. T., Sandoval, I. M., Zhang, Y., Li, T., Pittler, S. J., Chiu, W., Wensel, T. G. 2012; 151 (5): 1029–41

    Abstract

    Defects in primary cilia lead to devastating disease because of their roles in sensation and developmental signaling but much is unknown about ciliary structure and mechanisms of their formation and maintenance. We used cryo-electron tomography to obtain 3D maps of the connecting cilium and adjacent cellular structures of a modified primary cilium, the rod outer segment, from wild-type and genetically defective mice. The results reveal the molecular architecture of the cilium and provide insights into protein functions. They suggest that the ciliary rootlet is involved in cellular transport and stabilizes the axoneme. A defect in the BBSome membrane coat caused defects in vesicle targeting near the base of the cilium. Loss of the proteins encoded by the Cngb1 gene disrupted links between the disk and plasma membranes. The structures of the outer segment membranes support a model for disk morphogenesis in which basal disks are enveloped by the plasma membrane.

    View details for DOI 10.1016/j.cell.2012.10.038

    View details for PubMedID 23178122

    View details for PubMedCentralID PMC3582337

  • Direct electron detection yields cryo-EM reconstructions at resolutions beyond 3/4 Nyquist frequency. Journal of structural biology Bammes, B. E., Rochat, R. H., Jakana, J., Chen, D. H., Chiu, W. 2012; 177 (3): 589–601

    Abstract

    One limitation in electron cryo-microscopy (cryo-EM) is the inability to recover high-resolution signal from the image-recording media at the full-resolution limit of the transmission electron microscope. Direct electron detection using CMOS-based sensors for digitally recording images has the potential to alleviate this shortcoming. Here, we report a practical performance evaluation of a Direct Detection Device (DDD®) for biological cryo-EM at two different microscope voltages: 200 and 300 kV. Our DDD images of amorphous and graphitized carbon show strong per-pixel contrast with image resolution near the theoretical sampling limit of the data. Single-particle reconstructions of two frozen-hydrated bacteriophages, P22 and ε15, establish that the DDD is capable of recording usable signal for 3D reconstructions at about 4/5 of the Nyquist frequency, which is a vast improvement over the performance of conventional imaging media. We anticipate the unparalleled performance of this digital recording device will dramatically benefit cryo-EM for routine tomographic and single-particle structural determination of biological specimens.

    View details for DOI 10.1016/j.jsb.2012.01.008

    View details for PubMedID 22285189

    View details for PubMedCentralID PMC3314222

  • Reconstructing virus structures from nanometer to near-atomic resolutions with cryo-electron microscopy and tomography. Advances in experimental medicine and biology Chang, J., Liu, X., Rochat, R. H., Baker, M. L., Chiu, W. 2012; 726: 49–90

    Abstract

    The past few decades have seen tremendous advances in single-particle electron -cryo-microscopy (cryo-EM). The field has matured to the point that near-atomic resolution density maps can be generated for icosahedral viruses without the need for crystallization. In parallel, substantial progress has been made in determining the structures of nonicosahedrally arranged proteins in viruses by employing either single-particle cryo-EM or cryo-electron tomography (cryo-ET). Implicit in this course have been the availability of a new generation of electron cryo-microscopes and the development of the computational tools that are essential for generating these maps and models. This methodology has enabled structural biologists to analyze structures in increasing detail for virus particles that are in different morphogenetic states. Furthermore, electron imaging of frozen, hydrated cells, in the process of being infected by viruses, has also opened up a new avenue for studying virus structures "in situ". Here we present the common techniques used to acquire and process cryo-EM and cryo-ET data and discuss their implications for structural virology both now and in the future.

    View details for DOI 10.1007/978-1-4614-0980-9_4

    View details for PubMedID 22297510

    View details for PubMedCentralID PMC3954858

  • Constructing and validating initial Cα models from subnanometer resolution density maps with pathwalking. Structure (London, England : 1993) Baker, M. R., Rees, I., Ludtke, S. J., Chiu, W., Baker, M. L. 2012; 20 (3): 450–63

    Abstract

    A significant number of macromolecular structures solved by electron cryo-microscopy and X-ray crystallography obtain resolutions of 3.5-6Å, at which direct atomistic interpretation is difficult. To address this, we developed pathwalking, a semi-automated protocol to enumerate reasonable Cα models from near-atomic resolution density maps without a structural template or sequence-structure correspondence. Pathwalking uses an approach derived from the Traveling Salesman Problem to rapidly generate an ensemble of initial models for individual proteins, which can later be optimized to produce full atomic models. Pathwalking can also be used to validate and identify potential structural ambiguities in models generated from near-atomic resolution density maps. In this work, examples from the EMDB and PDB are used to assess the broad applicability and accuracy of our method. With the growing number of near-atomic resolution density maps from cryo-EM and X-ray crystallography, pathwalking can become an important tool in modeling protein structures.

    View details for DOI 10.1016/j.str.2012.01.008

    View details for PubMedID 22405004

    View details for PubMedCentralID PMC3307788

  • Gorgon and pathwalking: macromolecular modeling tools for subnanometer resolution density maps. Biopolymers Baker, M. L., Baker, M. R., Hryc, C. F., Ju, T., Chiu, W. 2012; 97 (9): 655–68

    Abstract

    The complex interplay of proteins and other molecules, often in the form of large transitory assemblies, are critical to cellular function. Today, X-ray crystallography and electron cryo-microscopy (cryo-EM) are routinely used to image these macromolecular complexes, though often at limited resolutions. Despite the rapidly growing number of macromolecular structures, few tools exist for modeling and annotating structures in the range of 3-10 Å resolution. To address this need, we have developed a number of utilities specifically targeting subnanometer resolution density maps. As part of the 2010 Cryo-EM Modeling Challenge, we demonstrated two of our latest de novo modeling tools, Pathwalking and Gorgon, as well as a tool for secondary structure identification (SSEHunter) and a new rigid-body/flexible fitting tool in Gorgon. In total, we submitted 30 structural models from ten different subnanometer resolution data sets in four of the six challenge categories. Each of our utlities produced accurate structural models and annotations across the various density maps. In the end, the utilities that we present here offer users a robust toolkit for analyzing and modeling protein structure in macromolecular assemblies at non-atomic resolutions.

    View details for DOI 10.1002/bip.22065

    View details for PubMedID 22696403

    View details for PubMedCentralID PMC3899894

  • Comparison of Segger and other methods for segmentation and rigid-body docking of molecular components in cryo-EM density maps. Biopolymers Pintilie, G., Chiu, W. 2012; 97 (9): 742–60

    Abstract

    Segmentation and docking are useful methods for the discovery of molecular components in electron cryo-microscopy (cryo-EM) density maps of macromolecular complexes. In this article, we describe the segmentation and docking methods implemented in Segger. For 11 targets posted in the 2010 cryo-EM challenge, we segmented the regions corresponding to individual molecular components using Segger. We then used the segmented regions to guide rigid-body docking of individual components. Docking results were evaluated by comparing the docked components with published structures, and by calculation of several scores, such as atom inclusion, density occupancy, and geometry clash. The accuracy of the component segmentation using Segger and other methods was assessed by comparing segmented regions with docked components.

    View details for DOI 10.1002/bip.22074

    View details for PubMedID 22696409

    View details for PubMedCentralID PMC3402182

  • A tail-like assembly at the portal vertex in intact herpes simplex type-1 virions. PLoS pathogens Schmid, M. F., Hecksel, C. W., Rochat, R. H., Bhella, D., Chiu, W., Rixon, F. J. 2012; 8 (10): e1002961

    Abstract

    Herpes viruses are prevalent and well characterized human pathogens. Despite extensive study, much remains to be learned about the structure of the genome packaging and release machinery in the capsids of these large and complex double-stranded DNA viruses. However, such machinery is well characterized in tailed bacteriophage, which share a common evolutionary origin with herpesvirus. In tailed bacteriophage, the genome exits from the virus particle through a portal and is transferred into the host cell by a complex apparatus (i.e. the tail) located at the portal vertex. Here we use electron cryo-tomography of human herpes simplex type-1 (HSV-1) virions to reveal a previously unsuspected feature at the portal vertex, which extends across the HSV-1 tegument layer to form a connection between the capsid and the viral membrane. The location of this assembly suggests that it plays a role in genome release into the nucleus and is also important for virion architecture.

    View details for DOI 10.1371/journal.ppat.1002961

    View details for PubMedID 23055933

    View details for PubMedCentralID PMC3464221

  • Cryo-EM Structure of a Group II Chaperonin in the Prehydrolysis ATP-Bound State Leading to Lid Closure STRUCTURE Zhang, J., Ma, B., DiMaio, F., Douglas, N. R., Joachimiak, L. A., Baker, D., Frydman, J., Levitt, M., Chiu, W. 2011; 19 (5): 633-639

    Abstract

    Chaperonins are large ATP-driven molecular machines that mediate cellular protein folding. Group II chaperonins use their "built-in lid" to close their central folding chamber. Here we report the structure of an archaeal group II chaperonin in its prehydrolysis ATP-bound state at subnanometer resolution using single particle cryo-electron microscopy (cryo-EM). Structural comparison of Mm-cpn in ATP-free, ATP-bound, and ATP-hydrolysis states reveals that ATP binding alone causes the chaperonin to close slightly with a ∼45° counterclockwise rotation of the apical domain. The subsequent ATP hydrolysis drives each subunit to rock toward the folding chamber and to close the lid completely. These motions are attributable to the local interactions of specific active site residues with the nucleotide, the tight couplings between the apical and intermediate domains within the subunit, and the aligned interactions between two subunits across the rings. This mechanism of structural changes in response to ATP is entirely different from those found in group I chaperonins.

    View details for DOI 10.1016/j.str.2011.03.005

    View details for Web of Science ID 000290815500006

    View details for PubMedID 21565698

  • Structural basis for scaffolding-mediated assembly and maturation of a dsDNA virus PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chen, D., Baker, M. L., Hryc, C. F., DiMaio, F., Jakana, J., Wu, W., Dougherty, M., Haase-Pettingell, C., Schmid, M. F., Jiang, W., Baker, D., King, J. A., Chiu, W. 2011; 108 (4): 1355-1360

    Abstract

    Formation of many dsDNA viruses begins with the assembly of a procapsid, containing scaffolding proteins and a multisubunit portal but lacking DNA, which matures into an infectious virion. This process, conserved among dsDNA viruses such as herpes viruses and bacteriophages, is key to forming infectious virions. Bacteriophage P22 has served as a model system for this study in the past several decades. However, how capsid assembly is initiated, where and how scaffolding proteins bind to coat proteins in the procapsid, and the conformational changes upon capsid maturation still remain elusive. Here, we report Cα backbone models for the P22 procapsid and infectious virion derived from electron cryomicroscopy density maps determined at 3.8- and 4.0-Å resolution, respectively, and the first procapsid structure at subnanometer resolution without imposing symmetry. The procapsid structures show the scaffolding protein interacting electrostatically with the N terminus (N arm) of the coat protein through its C-terminal helix-loop-helix motif, as well as unexpected interactions between 10 scaffolding proteins and the 12-fold portal located at a unique vertex. These suggest a critical role for the scaffolding proteins both in initiating the capsid assembly at the portal vertex and propagating its growth on a T = 7 icosahedral lattice. Comparison of the procapsid and the virion backbone models reveals coordinated and complex conformational changes. These structural observations allow us to propose a more detailed molecular mechanism for the scaffolding-mediated capsid assembly initiation including portal incorporation, release of scaffolding proteins upon DNA packaging, and maturation into infectious virions.

    View details for DOI 10.1073/pnas.1015739108

    View details for Web of Science ID 000286594800031

    View details for PubMedID 21220301

    View details for PubMedCentralID PMC3029737

  • Dual Action of ATP Hydrolysis Couples Lid Closure to Substrate Release into the Group II Chaperonin Chamber CELL Douglas, N. R., Reissmann, S., Zhang, J., Chen, B., Jakana, J., Kumar, R., Chiu, W., Frydman, J. 2011; 144 (2): 240-252

    Abstract

    Group II chaperonins are ATP-dependent ring-shaped complexes that bind nonnative polypeptides and facilitate protein folding in archaea and eukaryotes. A built-in lid encapsulates substrate proteins within the central chaperonin chamber. Here, we describe the fate of the substrate during the nucleotide cycle of group II chaperonins. The chaperonin substrate-binding sites are exposed, and the lid is open in both the ATP-free and ATP-bound prehydrolysis states. ATP hydrolysis has a dual function in the folding cycle, triggering both lid closure and substrate release into the central chamber. Notably, substrate release can occur in the absence of a lid, and lid closure can occur without substrate release. However, productive folding requires both events, so that the polypeptide is released into the confined space of the closed chamber where it folds. Our results show that ATP hydrolysis coordinates the structural and functional determinants that trigger productive folding.

    View details for DOI 10.1016/j.cell.2010.12.017

    View details for Web of Science ID 000286459900009

    View details for PubMedID 21241893

    View details for PubMedCentralID PMC3055171

  • Near-atomic-resolution cryo-EM for molecular virology. Current opinion in virology Hryc, C. F., Chen, D. H., Chiu, W. 2011; 1 (2): 110–17

    Abstract

    Electron cryo-microscopy (cryo-EM) is a technique in structural biology that is widely used to solve the three-dimensional structures of macromolecular assemblies, close to their biological and solution conditions. Recent improvements in cryo-EM and single-particle reconstruction methodologies have led to the determination of several virus structures at near-atomic resolution (3.3 - 4.6 Å). These cryo-EM structures not only resolve the Cα backbones and side-chain densities of viral capsid proteins, but also suggest functional roles that the protein domains and some key amino acid residues play. This paper reviews the recent advances in near-atomic-resolution cryo-EM for probing the mechanisms of virus assembly and morphogenesis.

    View details for DOI 10.1016/j.coviro.2011.05.019

    View details for PubMedID 21845206

    View details for PubMedCentralID PMC3155204

  • Modeling protein structure at near atomic resolutions with Gorgon. Journal of structural biology Baker, M. L., Abeysinghe, S. S., Schuh, S., Coleman, R. A., Abrams, A., Marsh, M. P., Hryc, C. F., Ruths, T., Chiu, W., Ju, T. 2011; 174 (2): 360–73

    Abstract

    Electron cryo-microscopy (cryo-EM) has played an increasingly important role in elucidating the structure and function of macromolecular assemblies in near native solution conditions. Typically, however, only non-atomic resolution reconstructions have been obtained for these large complexes, necessitating computational tools for integrating and extracting structural details. With recent advances in cryo-EM, maps at near-atomic resolutions have been achieved for several macromolecular assemblies from which models have been manually constructed. In this work, we describe a new interactive modeling toolkit called Gorgon targeted at intermediate to near-atomic resolution density maps (10-3.5 Å), particularly from cryo-EM. Gorgon's de novo modeling procedure couples sequence-based secondary structure prediction with feature detection and geometric modeling techniques to generate initial protein backbone models. Beyond model building, Gorgon is an extensible interactive visualization platform with a variety of computational tools for annotating a wide variety of 3D volumes. Examples from cryo-EM maps of Rotavirus and Rice Dwarf Virus are used to demonstrate its applicability to modeling protein structure.

    View details for DOI 10.1016/j.jsb.2011.01.015

    View details for PubMedID 21296162

    View details for PubMedCentralID PMC3078171

  • Workshop on the validation and modeling of electron cryo-microscopy structures of biological nanomachines. Pacific Symposium on Biocomputing. Pacific Symposium on Biocomputing Ludtke, S. J., Lawson, C. L., Kleywegt, G. J., Berman, H. M., Chiu, W. 2011: 369–73

    Abstract

    Electron cryo-microscopy (cryoEM) is a rapidly maturing methodology in structural biology, which now enables the determination of 3D structures of molecules, macromolecular complexes and cellular components at resolutions as high as 3.5Å, bridging the gap between light microscopy and X-ray crystallography/NMR. In recent years structures of many complex molecular machines have been visualized using this method. Single particle reconstruction, the most widely used technique in cryoEM, has recently demonstrated the capability of producing structures at resolutions approaching those of X-ray crystallography, with over a dozen structures at better than 5 Å resolution published to date. This method represents a significant new source of experimental data for molecular modeling and simulation studies. CryoEM derived maps and models are archived through EMDataBank.org joint deposition services to the EM Data Bank (EMDB) and Protein Data Bank (PDB), respectively. CryoEM maps are now being routinely produced over the 3 - 30 Å resolution range, and a number of computational groups are developing software for building coordinate models based on this data and developing validation techniques to better assess map and model accuracy. In this workshop we will present the results of the first cryoEM modeling challenge, in which computational groups were asked to apply their tools to a selected set of published cryoEM structures. We will also compare the results of the various applied methods, and discuss the current state of the art and how we can most productively move forward.

    View details for DOI 10.1142/9789814335058_0039

    View details for PubMedID 21121065

    View details for PubMedCentralID PMC3617577

  • Practical performance evaluation of a 10k × 10k CCD for electron cryo-microscopy. Journal of structural biology Bammes, B. E., Rochat, R. H., Jakana, J., Chiu, W. 2011; 175 (3): 384–93

    Abstract

    Electron cryo-microscopy (cryo-EM) images are commonly collected using either charge-coupled devices (CCD) or photographic film. Both film and the current generation of 16 megapixel (4k × 4k) CCD cameras have yielded high-resolution structures. Yet, despite the many advantages of CCD cameras, more than two times as many structures of biological macromolecules have been published in recent years using photographic film. The continued preference to film, especially for subnanometer-resolution structures, may be partially influenced by the finer sampling and larger effective specimen imaging area offered by film. Large format digital cameras may finally allow them to overtake film as the preferred detector for cryo-EM. We have evaluated a 111-megapixel (10k × 10k) CCD camera with a 9 μm pixel size. The spectral signal-to-noise ratios of low dose images of carbon film indicate that this detector is capable of providing signal up to at least 2/5 Nyquist frequency potentially retrievable for 3D reconstructions of biological specimens, resulting in more than double the effective specimen imaging area of existing 4k × 4k CCD cameras. We verified our estimates using frozen-hydrated ε15 bacteriophage as a biological test specimen with previously determined structure, yielding a ∼7 Å resolution single particle reconstruction from only 80 CCD frames. Finally, we explored the limits of current CCD technology by comparing the performance of this detector to various CCD cameras used for recording data yielding subnanometer resolution cryo-EM structures submitted to the electron microscopy data bank (http://www.emdatabank.org/).

    View details for DOI 10.1016/j.jsb.2011.05.012

    View details for PubMedID 21619932

    View details for PubMedCentralID PMC3150461

  • Structure of Trypanosoma brucei flagellum accounts for its bihelical motion. Proceedings of the National Academy of Sciences of the United States of America Koyfman, A. Y., Schmid, M. F., Gheiratmand, L., Fu, C. J., Khant, H. A., Huang, D., He, C. Y., Chiu, W. 2011; 108 (27): 11105–8

    Abstract

    Trypanosoma brucei is a parasitic protozoan that causes African sleeping sickness. It contains a flagellum required for locomotion and viability. In addition to a microtubular axoneme, the flagellum contains a crystalline paraflagellar rod (PFR) and connecting proteins. We show here, by cryoelectron tomography, the structure of the flagellum in three bending states. The PFR lattice in straight flagella repeats every 56 nm along the length of the axoneme, matching the spacing of the connecting proteins. During flagellar bending, the PFR crystallographic unit cell lengths remain constant while the interaxial angles vary, similar to a jackscrew. The axoneme drives the expansion and compression of the PFR lattice. We propose that the PFR modifies the in-plane axoneme motion to produce the characteristic trypanosome bihelical motility as captured by high-speed light microscope videography.

    View details for DOI 10.1073/pnas.1103634108

    View details for PubMedID 21690369

    View details for PubMedCentralID PMC3131312

  • Flexible architecture of IP3R1 by Cryo-EM. Structure (London, England : 1993) Ludtke, S. J., Tran, T. P., Ngo, Q. T., Moiseenkova-Bell, V. Y., Chiu, W., Serysheva, I. I. 2011; 19 (8): 1192–99

    Abstract

    Inositol 1,4,5-trisphosphate receptors (IP3Rs) play a fundamental role in generating Ca2+ signals that trigger many cellular processes in virtually all eukaryotic cells. Thus far, the three-dimensional (3D) structure of these channels has remained extremely controversial. Here, we report a subnanometer resolution electron cryomicroscopy (cryo-EM) structure of a fully functional type 1 IP3R from cerebellum in the closed state. The transmembrane region reveals a twisted bundle of four α helices, one from each subunit, that form a funnel shaped structure around the 4-fold symmetry axis, strikingly similar to the ion-conduction pore of K+ channels. The lumenal face of IP3R1 has prominent densities that surround the pore entrance and similar to the highly structured turrets of Kir channels. 3D statistical analysis of the cryo-EM density map identifies high variance in the cytoplasmic region. This structural variation could be attributed to genuine structural flexibility of IP3R1.

    View details for DOI 10.1016/j.str.2011.05.003

    View details for PubMedID 21827954

    View details for PubMedCentralID PMC3154621

  • 4.4 Å cryo-EM structure of an enveloped alphavirus Venezuelan equine encephalitis virus. The EMBO journal Zhang, R., Hryc, C. F., Cong, Y., Liu, X., Jakana, J., Gorchakov, R., Baker, M. L., Weaver, S. C., Chiu, W. 2011; 30 (18): 3854–63

    Abstract

    Venezuelan equine encephalitis virus (VEEV), a member of the membrane-containing Alphavirus genus, is a human and equine pathogen, and has been developed as a biological weapon. Using electron cryo-microscopy (cryo-EM), we determined the structure of an attenuated vaccine strain, TC-83, of VEEV to 4.4 Å resolution. Our density map clearly resolves regions (including E1, E2 transmembrane helices and cytoplasmic tails) that were missing in the crystal structures of domains of alphavirus subunits. These new features are implicated in the fusion, assembly and budding processes of alphaviruses. Furthermore, our map reveals the unexpected E3 protein, which is cleaved and generally thought to be absent in the mature VEEV. Our structural results suggest a mechanism for the initial stage of nucleocapsid core formation, and shed light on the virulence attenuation, host recognition and neutralizing activities of VEEV and other alphavirus pathogens.

    View details for DOI 10.1038/emboj.2011.261

    View details for PubMedID 21829169

    View details for PubMedCentralID PMC3173789

  • MOTIF-EM: an automated computational tool for identifying conserved regions in CryoEM structures BIOINFORMATICS Saha, M., Levitt, M., Chiu, W. 2010; 26 (12): i301-i309

    Abstract

    We present a new, first-of-its-kind, fully automated computational tool MOTIF-EM for identifying regions or domains or motifs in cryoEM maps of large macromolecular assemblies (such as chaperonins, viruses, etc.) that remain conformationally conserved. As a by-product, regions in structures that are not conserved are revealed: this can indicate local molecular flexibility related to biological activity. MOTIF-EM takes cryoEM volumetric maps as inputs. The technique used by MOTIF-EM to detect conserved sub-structures is inspired by a recent breakthrough in 2D object recognition. The technique works by constructing rotationally invariant, low-dimensional representations of local regions in the input cryoEM maps. Correspondences are established between the reduced representations (by comparing them using a simple metric) across the input maps. The correspondences are clustered using hash tables and graph theory is used to retrieve conserved structural domains or motifs. MOTIF-EM has been used to extract conserved domains occurring in large macromolecular assembly maps, including as those of viruses P22 and epsilon 15, Ribosome 70S, GroEL, that remain structurally conserved in different functional states. Our method can also been used to build atomic models for some maps. We also used MOTIF-EM to identify the conserved folds shared among dsDNA bacteriophages HK97, Epsilon 15, and ô29, though they have low-sequence similarity. Supplementary information: Supplementary data are available at Bioinformatics online.

    View details for DOI 10.1093/bioinformatics/btq195

    View details for Web of Science ID 000278689000037

    View details for PubMedID 20529921

    View details for PubMedCentralID PMC2881380

  • 4.0 angstrom Resolution Cryo-EM Structure of the Mammalian Chaperonin TRiC/CCT Reveals its Unique Subunit Arrangement Cong, Y., Baker, M. L., Jakana, J., Woolford, D., Miller, E. J., Reissmann, S., Kumar, R. N., Redding-Johanson, A. M., Batth, T. S., Mukhopadhyay, A., Ludtke, S. J., Frydman, J., Chiu, W. FEDERATION AMER SOC EXP BIOL. 2010
  • 4.0-angstrom resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cong, Y., Baker, M. L., Jakana, J., Woolford, D., Miller, E. J., Reissmann, S., Kumar, R. N., Redding-Johanson, A. M., Batth, T. S., Mukhopadhyay, A., Ludtke, S. J., Frydman, J., Chiu, W. 2010; 107 (11): 4967-4972

    Abstract

    The essential double-ring eukaryotic chaperonin TRiC/CCT (TCP1-ring complex or chaperonin containing TCP1) assists the folding of approximately 5-10% of the cellular proteome. Many TRiC substrates cannot be folded by other chaperonins from prokaryotes or archaea. These unique folding properties are likely linked to TRiC's unique heterooligomeric subunit organization, whereby each ring consists of eight different paralogous subunits in an arrangement that remains uncertain. Using single particle cryo-EM without imposing symmetry, we determined the mammalian TRiC structure at 4.7-A resolution. This revealed the existence of a 2-fold axis between its two rings resulting in two homotypic subunit interactions across the rings. A subsequent 2-fold symmetrized map yielded a 4.0-A resolution structure that evinces the densities of a large fraction of side chains, loops, and insertions. These features permitted unambiguous identification of all eight individual subunits, despite their sequence similarity. Independent biochemical near-neighbor analysis supports our cryo-EM derived TRiC subunit arrangement. We obtained a Calpha backbone model for each subunit from an initial homology model refined against the cryo-EM density. A subsequently optimized atomic model for a subunit showed approximately 95% of the main chain dihedral angles in the allowable regions of the Ramachandran plot. The determination of the TRiC subunit arrangement opens the way to understand its unique function and mechanism. In particular, an unevenly distributed positively charged wall lining the closed folding chamber of TRiC differs strikingly from that of prokaryotic and archaeal chaperonins. These interior surface chemical properties likely play an important role in TRiC's cellular substrate specificity.

    View details for DOI 10.1073/pnas.0913774107

    View details for Web of Science ID 000275714300032

    View details for PubMedID 20194787

    View details for PubMedCentralID PMC2841888

  • 4.0 angstrom Cryo-EM Structure of the Mammalian Chaperonin: TRiC/CCT Cong, Y., Baker, M., Jakana, J., Woolford, D., Reissmann, S., Ludtke, S. J., Frydman, J., Chiu, W. CELL PRESS. 2010: 222A–223A
  • Radiation damage effects at four specimen temperatures from 4 to 100 K. Journal of structural biology Bammes, B. E., Jakana, J., Schmid, M. F., Chiu, W. 2010; 169 (3): 331–41

    Abstract

    Radiation damage is the primary factor that limits resolution in electron cryo-microscopy (cryo-EM) of frozen-hydrated biological samples. Negative effects of radiation damage are attenuated by cooling specimens to cryogenic temperatures using liquid nitrogen or liquid helium. We have examined the relationship between specimen temperature and radiation damage across a broad spectrum of resolution by analyzing images of frozen-hydrated catalase crystal at four specimen temperatures: 4, 25, 42, and 100K. For each temperature, "exposure series" were collected consisting of consecutive images of the same area of sample, each with 10 e(-)/A(2) exposure per image. Radiation damage effects were evaluated by examining the correlation between cumulative exposure and normalized amplitudes or IQ values of Bragg peaks across a broad range of resolution (4.0-173.5A). Results indicate that for sub-nanometer resolution, liquid nitrogen specimen temperature (100K) provides the most consistent high-quality data while yielding statistically equivalent protection from radiation damage compared to the three lower temperatures. At lower resolution, suitable for tomography, intermediate temperatures (25 or 42K) may provide a modest improvement in cryo-protection without introducing deleterious effects evident at 4 K.

    View details for DOI 10.1016/j.jsb.2009.11.001

    View details for PubMedID 19903530

    View details for PubMedCentralID PMC2826528

  • Structural changes in a marine podovirus associated with release of its genome into Prochlorococcus. Nature structural & molecular biology Liu, X., Zhang, Q., Murata, K., Baker, M. L., Sullivan, M. B., Fu, C., Dougherty, M. T., Schmid, M. F., Osburne, M. S., Chisholm, S. W., Chiu, W. 2010; 17 (7): 830–36

    Abstract

    Podovirus P-SSP7 infects Prochlorococcus marinus, the most abundant oceanic photosynthetic microorganism. Single-particle cryo-electron microscopy yields icosahedral and asymmetrical structures of infectious P-SSP7 with 4.6-A and 9-A resolution, respectively. The asymmetric reconstruction reveals how symmetry mismatches are accommodated among five of the gene products at the portal vertex. Reconstructions of infectious and empty particles show a conformational change of the 'valve' density in the nozzle, an orientation difference in the tail fibers, a disordering of the C terminus of the portal protein and the disappearance of the core proteins. In addition, cryo-electron tomography of P-SSP7 infecting Prochlorococcus showed the same tail-fiber conformation as that in empty particles. Our observations suggest a mechanism whereby, upon binding to the host cell, the tail fibers induce a cascade of structural alterations of the portal vertex complex that triggers DNA release.

    View details for DOI 10.1038/nsmb.1823

    View details for PubMedID 20543830

    View details for PubMedCentralID PMC2924429

  • Zernike phase contrast cryo-electron microscopy and tomography for structure determination at nanometer and subnanometer resolutions. Structure (London, England : 1993) Murata, K., Liu, X., Danev, R., Jakana, J., Schmid, M. F., King, J., Nagayama, K., Chiu, W. 2010; 18 (8): 903–12

    Abstract

    Zernike phase contrast cryo-electron microscopy (ZPC-cryoEM) is an emerging technique that is capable of producing higher image contrast than conventional cryoEM. By combining this technique with advanced image processing methods, we achieved subnanometer resolution for two biological specimens: 2D bacteriorhodopsin crystal and epsilon15 bacteriophage. For an asymmetric reconstruction of epsilon15 bacteriophage, ZPC-cryoEM can reduce the required amount of data by a factor of approximately 3, compared with conventional cryoEM. The reconstruction was carried out to 13 A resolution without the need to correct the contrast transfer function. New structural features at the portal vertex of the epsilon15 bacteriophage are revealed in this reconstruction. Using ZPC cryo-electron tomography (ZPC-cryoET), a similar level of data reduction and higher resolution structures of epsilon15 bacteriophage can be obtained relative to conventional cryoET. These results show quantitatively the benefits of ZPC-cryoEM and ZPC-cryoET for structural determinations of macromolecular machines at nanometer and subnanometer resolutions.

    View details for DOI 10.1016/j.str.2010.06.006

    View details for PubMedID 20696391

    View details for PubMedCentralID PMC2925294

  • Visualizing the structural changes of bacteriophage Epsilon15 and its Salmonella host during infection. Journal of molecular biology Chang, J. T., Schmid, M. F., Haase-Pettingell, C., Weigele, P. R., King, J. A., Chiu, W. 2010; 402 (4): 731–40

    Abstract

    The efficient mechanism by which double-stranded DNA bacteriophages deliver their chromosome across the outer membrane, cell wall, and inner membrane of Gram-negative bacteria remains obscure. Advances in single-particle electron cryomicroscopy have recently revealed details of the organization of the DNA injection apparatus within the mature virion for various bacteriophages, including epsilon15 (ɛ15) and P-SSP7. We have used electron cryotomography and three-dimensional subvolume averaging to capture snapshots of ɛ15 infecting its host Salmonella anatum. These structures suggest the following stages of infection. In the first stage, the tailspikes of ɛ15 attach to the surface of the host cell. Next, ɛ15's tail hub attaches to a putative cell receptor and establishes a tunnel through which the injection core proteins behind the portal exit the virion. A tube spanning the periplasmic space is formed for viral DNA passage, presumably from the rearrangement of core proteins or from cellular components. This tube would direct the DNA into the cytoplasm and protect it from periplasmic nucleases. Once the DNA has been injected into the cell, the tube and portal seals, and the empty bacteriophage remains at the cell surface.

    View details for DOI 10.1016/j.jmb.2010.07.058

    View details for PubMedID 20709082

    View details for PubMedCentralID PMC3164490

  • Workshop on molecular animation. Structure (London, England : 1993) Bromberg, S., Chiu, W., Ferrin, T. E. 2010; 18 (10): 1261–65

    Abstract

    From February 25 to 26, 2010, in San Francisco, the Resource for Biocomputing, Visualization, and Informatics (RBVI) and the National Center for Macromolecular Imaging (NCMI) hosted a molecular animation workshop for 21 structural biologists, molecular animators, and creators of molecular visualization software. Molecular animation aims to visualize scientific understanding of biomolecular processes and structures. The primary goal of the workshop was to identify the necessary tools for producing high-quality molecular animations, understanding complex molecular and cellular structures, creating publication supplementary materials and conference presentations, and teaching science to students and the public. Another use of molecular animation emerged in the workshop: helping to focus scientific inquiry about the motions of molecules and enhancing informal communication within and between laboratories.

    View details for DOI 10.1016/j.str.2010.09.001

    View details for PubMedID 20947014

    View details for PubMedCentralID PMC3071847

  • Cryo-EM of macromolecular assemblies at near-atomic resolution NATURE PROTOCOLS Baker, M. L., Zhang, J., Ludtke, S. J., Chiu, W. 2010; 5 (10): 1697-1708

    Abstract

    With single-particle electron cryomicroscopy (cryo-EM), it is possible to visualize large, macromolecular assemblies in near-native states. Although subnanometer resolutions have been routinely achieved for many specimens, state of the art cryo-EM has pushed to near-atomic (3.3-4.6 Å) resolutions. At these resolutions, it is now possible to construct reliable atomic models directly from the cryo-EM density map. In this study, we describe our recently developed protocols for performing the three-dimensional reconstruction and modeling of Mm-cpn, a group II chaperonin, determined to 4.3 Å resolution. This protocol, utilizing the software tools EMAN, Gorgon and Coot, can be adapted for use with nearly all specimens imaged with cryo-EM that target beyond 5 Å resolution. Additionally, the feature recognition and computational modeling tools can be applied to any near-atomic resolution density maps, including those from X-ray crystallography.

    View details for DOI 10.1038/nprot.2010.126

    View details for Web of Science ID 000282369100010

    View details for PubMedID 20885381

    View details for PubMedCentralID PMC3107675

  • Rocking Motion of a Protein-Folding Nano-Machine Revealed By Single-Particle Cryo-Em Zhang, J., Baker, M. L., Schroeder, G., Douglas, N. R., Jakana, J., Fu, C. J., Levitt, M., Ludtke, S. J., Frydman, J., Chiu, W. CELL PRESS. 2010: 33A
  • Conformational Change of a Group II Chaperonin in Different States Revealed by Single-particle Cryo-EM Zhang, J., Baker, M. L., Schroeder, G., Douglas, N. R., Jakana, J., Fu, C. J., Levitt, M., Ludtke, S. J., Frydman, J., Chiu, W. ADENINE PRESS. 2009: 844–44
  • Rocking Motion of the Equatorial Domains of a Group II Chaperonin between Two Biochemical States Revealed by Single-Particle Cryo-EM at Near-atomic and Subnanometer Resolutions Zhang, J., Baker, M., Schroeder, G., Douglas, N., Jakana, J., Fu, C., Levitt, M., Ludtke, S., Frydman, J., Chiu, W. FEDERATION AMER SOC EXP BIOL. 2009
  • Conformational Changes of Eukaryotic Chaperonin TRiC/CCT in the Nucleotide Cycle Revealed by CryoEM Cong, Y., Schroeder, G. F., Jakana, J., Reissmann, S., Levitt, M., Ludtke, S. J., Frydman, J., Chiu, W. FEDERATION AMER SOC EXP BIOL. 2009
  • Estimating contrast transfer function and associated parameters by constrained non-linear optimization JOURNAL OF MICROSCOPY Yang, C., Jiang, W., Chen, D., Adiga, U., Ng, E. G., Chiu, W. 2009; 233 (3): 391-403

    Abstract

    The three-dimensional reconstruction of macromolecules from two-dimensional single-particle electron images requires determination and correction of the contrast transfer function (CTF) and envelope function. A computational algorithm based on constrained non-linear optimization is developed to estimate the essential parameters in the CTF and envelope function model simultaneously and automatically. The application of this estimation method is demonstrated with focal series images of amorphous carbon film as well as images of ice-embedded icosahedral virus particles suspended across holes.

    View details for DOI 10.1111/j.1365-2818.2009.03137.x

    View details for Web of Science ID 000263758000005

    View details for PubMedID 19250460

  • JADAS: A customizable automated data acquisition system and its application to ice-embedded single particles JOURNAL OF STRUCTURAL BIOLOGY Zhang, J., Nakamura, N., Shimizu, Y., Liang, N., Liu, X., Jakana, J., Marsh, M. P., Booth, C. R., Shinkawa, T., Nakata, M., Chiu, W. 2009; 165 (1): 1-9

    Abstract

    The JEOL Automated Data Acquisition System (JADAS) is a software system built for the latest generation of the JEOL Transmission Electron Microscopes. It is designed to partially or fully automate image acquisition for ice-embedded single particles under low dose conditions. Its built-in flexibility permits users to customize the order of various imaging operations. In this paper, we describe how JADAS is used to accurately locate and image suitable specimen areas on a grid of ice-embedded particles. We also demonstrate the utility of JADAS by imaging the epsilon 15 bacteriophage with the JEM3200FSC electron cryo-microscope, showing that sufficient images can be collected in a single 8h session to yield a subnanometer resolution structure which agrees with the previously determined structure.

    View details for DOI 10.1016/j.jsb.2008.09.006

    View details for Web of Science ID 000262448400001

    View details for PubMedID 18926912

    View details for PubMedCentralID PMC2634810

  • Interprotofilament interactions between Alzheimer's Abeta1-42 peptides in amyloid fibrils revealed by cryoEM. Proceedings of the National Academy of Sciences of the United States of America Zhang, R., Hu, X., Khant, H., Ludtke, S. J., Chiu, W., Schmid, M. F., Frieden, C., Lee, J. M. 2009; 106 (12): 4653–58

    Abstract

    Alzheimer's disease is a neurodegenerative disorder characterized by the accumulation of amyloid plaques in the brain. This amyloid primarily contains amyloid-beta (Abeta), a 39- to 43-aa peptide derived from the proteolytic cleavage of the endogenous amyloid precursor protein. The 42-residue-length Abeta peptide (Abeta(1-42)), the most abundant Abeta peptide found in plaques, has a much greater propensity to self-aggregate into fibrils than the other peptides and is believed to be more pathogenic. Synthetic human Abeta(1-42) peptides self-aggregate into stable but poorly-ordered helical filaments. We determined their structure to approximately 10-A resolution by using cryoEM and the iterative real-space reconstruction method. This structure reveals 2 protofilaments winding around a hollow core. Previous hairpin-like NMR models for Abeta(17-42) fit well in the cryoEM density map and reveal that the juxtaposed protofilaments are joined via the N terminus of the peptide from 1 protofilament connecting to the loop region of the peptide in the opposite protofilament. This model of mature Abeta(1-42) fibrils is markedly different from previous cryoEM models of Abeta(1-40) fibrils. In our model, the C terminus of Abeta forms the inside wall of the hollow core, which is supported by partial proteolysis analysis.

    View details for DOI 10.1073/pnas.0901085106

    View details for PubMedID 19264960

    View details for PubMedCentralID PMC2660777

  • Structural mechanism of SDS-induced enzyme activity of scorpion hemocyanin revealed by electron cryomicroscopy. Structure (London, England : 1993) Cong, Y., Zhang, Q., Woolford, D., Schweikardt, T., Khant, H., Dougherty, M., Ludtke, S. J., Chiu, W., Decker, H. 2009; 17 (5): 749–58

    Abstract

    Phenoloxidases (POs) occur in all organisms and are involved in skin and hair coloring in mammals, and initiating melanization in wound healing. Mutation or overexpression of PO can cause albinism or melanoma, respectively. SDS can convert inactive PO and the oxygen carrier hemocyanin (Hc) into enzymatically active PO. Here we present single-particle cryo-EM maps at subnanometer resolution and pseudoatomic models of the 24-oligomeric Hc from scorpion Pandinus imperator in resting and SDS-activated states. Our structural analyses led to a plausible mechanism of Hc enzyme PO activation: upon SDS activation, the intrinsically flexible Hc domain I twists away from domains II and III in each subunit, exposing the entrance to the active site; this movement is stabilized by enhanced interhexamer and interdodecamer interactions, particularly in the central linker subunits. This mechanism could be applicable to other type 3 copper proteins, as the active site is highly conserved.

    View details for DOI 10.1016/j.str.2009.03.005

    View details for PubMedID 19446530

    View details for PubMedCentralID PMC2705691

  • Location and flexibility of the unique C-terminal tail of Aquifex aeolicus co-chaperonin protein 10 as derived by cryo-electron microscopy and biophysical techniques JOURNAL OF MOLECULAR BIOLOGY Chen, D., Luke, K., Zhang, J., Chiu, W., Wittung-Stafshede, P. 2008; 381 (3): 707-717

    Abstract

    Co-chaperonin protein 10 (cpn10, GroES in Escherichia coli) is a ring-shaped heptameric protein that facilitates substrate folding when in complex with cpn60 (GroEL in E. coli). The cpn10 from the hyperthermophilic, ancient bacterium Aquifex aeolicus (Aacpn10) has a 25-residue C-terminal extension in each monomer not found in any other cpn10 protein. Earlier in vitro work has shown that this tail is not needed for heptamer assembly or protein function. Without the tail, however, the heptamers (Aacpn10del-25) readily aggregate into fibrillar stacked rings. To explain this phenomenon, we performed binding experiments with a peptide construct of the tail to establish its specificity for Aacpn10del-25 and used cryo-electron microscopy to determine the three-dimensional (3D) structure of the GroEL-Aacpn10-ADP complex at an 8-A resolution. We found that the GroEL-Aacpn10 structure is similar to the GroEL-GroES structure at this resolution, suggesting that Aacpn10 has molecular interactions with cpn60 similar to other cpn10s. The cryo-electron microscopy density map does not directly reveal the density of the Aacpn10 25-residue tail. However, the 3D statistical variance coefficient map computed from multiple 3D reconstructions with randomly selected particle images suggests that the tail is located at the Aacpn10 monomer-monomer interface and extends toward the cis-ring apical domain of GroEL. The tail at this location does not block the formation of a functional co-chaperonin/chaperonin complex but limits self-aggregation into linear fibrils at high temperatures. In addition, the 3D variance coefficient map identifies several regions inside the GroEL-Aacpn10 complex that have flexible conformations. This observation is in full agreement with the structural properties of an effective chaperonin.

    View details for DOI 10.1016/j.jmb.2008.06.021

    View details for Web of Science ID 000258736700018

    View details for PubMedID 18588898

    View details for PubMedCentralID PMC2612737

  • Mechanism of lid closure in the eukaryotic chaperonin TRiC/CCT NATURE STRUCTURAL & MOLECULAR BIOLOGY Booth, C. R., Meyer, A. S., Cong, Y., Topf, M., Sali, A., Ludtke, S. J., Chiu, W., Frydman, J. 2008; 15 (7): 746-753

    Abstract

    All chaperonins mediate ATP-dependent polypeptide folding by confining substrates within a central chamber. Intriguingly, the eukaryotic chaperonin TRiC (also called CCT) uses a built-in lid to close the chamber, whereas prokaryotic chaperonins use a detachable lid. Here we determine the mechanism of lid closure in TRiC using single-particle cryo-EM and comparative protein modeling. Comparison of TRiC in its open, nucleotide-free, and closed, nucleotide-induced states reveals that the interdomain motions leading to lid closure in TRiC are radically different from those of prokaryotic chaperonins, despite their overall structural similarity. We propose that domain movements in TRiC are coordinated through unique interdomain contacts within each subunit and, further, these contacts are absent in prokaryotic chaperonins. Our findings show how different mechanical switches can evolve from a common structural framework through modification of allosteric networks.

    View details for DOI 10.1038/nsmb.1436

    View details for Web of Science ID 000257412500018

    View details for PubMedID 18536725

    View details for PubMedCentralID PMC2546500

  • Subnanometer-resolution electron cryomicroscopy-based domain models for the cytoplasmic region of skeletal muscle RyR channel. Proceedings of the National Academy of Sciences of the United States of America Serysheva, I. I., Ludtke, S. J., Baker, M. L., Cong, Y., Topf, M., Eramian, D., Sali, A., Hamilton, S. L., Chiu, W. 2008; 105 (28): 9610–15

    Abstract

    The skeletal muscle Ca(2+) release channel (RyR1), a homotetramer, regulates the release of Ca(2+) from the sarcoplasmic reticulum to initiate muscle contraction. In this work, we have delineated the RyR1 monomer boundaries in a subnanometer-resolution electron cryomicroscopy (cryo-EM) density map. In the cytoplasmic region of each RyR1 monomer, 36 alpha-helices and 7 beta-sheets can be resolved. A beta-sheet was also identified close to the membrane-spanning region that resembles the cytoplasmic pore structures of inward rectifier K(+) channels. Three structural folds, generated for amino acids 12-565 using comparative modeling and cryo-EM density fitting, localize close to regions implicated in communication with the voltage sensor in the transverse tubules. Eleven of the 15 disease-related residues for these domains are mapped to the surface of these models. Four disease-related residues are found in a basin at the interfaces of these regions, creating a pocket in which the immunophilin FKBP12 can fit. Taken together, these results provide a structural context for both channel gating and the consequences of certain malignant hyperthermia and central core disease-associated mutations in RyR1.

    View details for DOI 10.1073/pnas.0803189105

    View details for PubMedID 18621707

    View details for PubMedCentralID PMC2474495

  • Crystallographic conformers of actin in a biologically active bundle of filaments. Journal of molecular biology Cong, Y., Topf, M., Sali, A., Matsudaira, P., Dougherty, M., Chiu, W., Schmid, M. F. 2008; 375 (2): 331–36

    Abstract

    Actin carries out many of its cellular functions through its filamentous form; thus, understanding the detailed structure of actin filaments is an essential step in achieving a mechanistic understanding of actin function. The acrosomal bundle in the Limulus sperm has been shown to be a quasi-crystalline array with an asymmetric unit composed of a filament with 14 actin-scruin pairs. The bundle in its true discharge state penetrates the jelly coat of the egg. Our previous electron crystallographic reconstruction demonstrated that the actin filament cross-linked by scruin in this acrosomal bundle state deviates significantly from a perfect F-actin helix. In that study, the tertiary structure of each of the 14 actin protomers in the asymmetric unit of the bundle filament was assumed to be constant. In the current study, an actin filament atomic model in the acrosomal bundle has been refined by combining rigid-body docking with multiple actin crystal structures from the Protein Data Bank and constrained energy minimization. Our observation demonstrates that actin protomers adopt different tertiary conformations when they form an actin filament in the bundle. The scruin and bundle packing forces appear to influence the tertiary and quaternary conformations of actin in the filament of this biologically active bundle.

    View details for DOI 10.1016/j.jmb.2007.10.027

    View details for PubMedID 18022194

    View details for PubMedCentralID PMC2680129

  • De novo backbone trace of GroEL from single particle electron cryomicroscopy. Structure (London, England : 1993) Ludtke, S. J., Baker, M. L., Chen, D. H., Song, J. L., Chuang, D. T., Chiu, W. 2008; 16 (3): 441–48

    Abstract

    In this work, we employ single-particle electron cryo-microscopy (cryo-EM) to reconstruct GroEL to approximately 4 A resolution with both D7 and C7 symmetry. Using a newly developed skeletonization algorithm and secondary structure element identification in combination with sequence-based secondary structure prediction, we demonstrate that it is possible to achieve a de novo Calpha trace directly from a cryo-EM reconstruction. The topology of our backbone trace is completely accurate, though subtle alterations illustrate significant differences from existing crystal structures. In the map with C7 symmetry, the seven monomers in each ring are identical; however, the subunits have a subtly different structure in each ring, particularly in the equatorial domain. These differences include an asymmetric salt bridge, density in the nucleotide-binding pocket of only one ring, and small shifts in alpha helix positions. This asymmetric conformation is different from previous asymmetric structures, including GroES-bound GroEL, and may represent a "primed state" in the chaperonin pathway.

    View details for DOI 10.1016/j.str.2008.02.007

    View details for PubMedID 18334219

  • Achievable resolution from images of biological specimens acquired from a 4k x 4k CCD camera in a 300-kV electron cryomicroscope. Journal of structural biology Chen, D. H., Jakana, J., Liu, X., Schmid, M. F., Chiu, W. 2008; 163 (1): 45–52

    Abstract

    Bacteriorhodopsin and epsilon 15 bacteriophage were used as biological test specimens to evaluate the potential structural resolution with images captured from a 4k x 4k charge-coupled device (CCD) camera in a 300-kV electron cryomicroscope. The phase residuals computed from the bacteriorhodopsin CCD images taken at 84,000x effective magnification averaged 15.7 degrees out to 5.8-A resolution relative to Henderson's published values. Using a single-particle reconstruction technique, we obtained an 8.2-A icosahedral structure of epsilon 15 bacteriophage with the CCD images collected at an effective magnification of 56,000x. These results demonstrate that it is feasible to retrieve biological structures to a resolution close to 2/3 of the Nyquist frequency from the CCD images recorded in a 300-kV electron cryomicroscope at a moderately high but practically acceptable microscope magnification.

    View details for DOI 10.1016/j.jsb.2008.04.001

    View details for PubMedID 18514542

    View details for PubMedCentralID PMC2504495

  • Essential function of the built-in lid in the allosteric regulation of eukaryotic and archaeal chaperonins NATURE STRUCTURAL & MOLECULAR BIOLOGY Reissmann, S., Parnot, C., Booth, C. R., Chiu, W., Frydman, J. 2007; 14 (5): 432-440

    Abstract

    Chaperonins are allosteric double-ring ATPases that mediate cellular protein folding. ATP binding and hydrolysis control opening and closing of the central chaperonin chamber, which transiently provides a protected environment for protein folding. During evolution, two strategies to close the chaperonin chamber have emerged. Archaeal and eukaryotic group II chaperonins contain a built-in lid, whereas bacterial chaperonins use a ring-shaped cofactor as a detachable lid. Here we show that the built-in lid is an allosteric regulator of group II chaperonins, which helps synchronize the subunits within one ring and, to our surprise, also influences inter-ring communication. The lid is dispensable for substrate binding and ATP hydrolysis, but is required for productive substrate folding. These regulatory functions of the lid may serve to allow the symmetrical chaperonins to function as 'two-stroke' motors and may also provide a timer for substrate encapsulation within the closed chamber.

    View details for DOI 10.1038/nsmb1236

    View details for Web of Science ID 000246187400017

    View details for PubMedID 17460696

    View details for PubMedCentralID PMC3339572

  • Modular software platform for low-dose electron microscopy and tomography. Journal of microscopy Marsh, M. P., Chang, J. T., Booth, C. R., Liang, N. L., Schmid, M. F., Chiu, W. 2007; 228 (Pt 3): 384–89

    Abstract

    Transmission electron microscopy imaging protocols required by structural scientists vary widely and can be laborious without tailor-made applications. We present here the jeol automated microscopy expert system (james) api integrator, a programming library for computer control of transmission electron microscopy operations and equipment. james has been implemented on JEOL microscopes with Gatan CCDs but is designed to be modular so it can be adapted to run on different microscopes and detectors. We have used the james api integrator to develop two applications for low-dose digital imaging: james imaging application and the mr t tomographic imaging application. Both applications have been widely used within our NCRR-supported Center for routine data collection and are now made available for public download.

    View details for DOI 10.1111/j.1365-2818.2007.01856.x

    View details for PubMedID 18045333

    View details for PubMedCentralID PMC4384816

  • Electron cryotomography reveals the portal in the herpesvirus capsid. Journal of virology Chang, J. T., Schmid, M. F., Rixon, F. J., Chiu, W. 2007; 81 (4): 2065–68

    Abstract

    Herpes simplex virus type 1 is a human pathogen responsible for a range of illnesses from cold sores to encephalitis. The icosahedral capsid has a portal at one fivefold vertex which, by analogy to portal-containing phages, is believed to mediate genome entry and exit. We used electron cryotomography to determine the structure of capsids lacking pentons. The portal vertex appears different from pentons, being located partially inside the capsid shell, a position equivalent to that of bacteriophage portals. Such similarity in portal organization supports the idea of the evolutionary relatedness of these viruses.

    View details for DOI 10.1128/JVI.02053-06

    View details for PubMedID 17151101

    View details for PubMedCentralID PMC1797573

  • Identification of secondary structure elements in intermediate-resolution density maps. Structure (London, England : 1993) Baker, M. L., Ju, T., Chiu, W. 2007; 15 (1): 7–19

    Abstract

    An increasing number of structural studies of large macromolecular complexes, both in X-ray crystallography and cryo-electron microscopy, have resulted in intermediate-resolution (5-10 A) density maps. Despite being limited in resolution, significant structural and functional information may be extractable from these maps. To aid in the analysis and annotation of these complexes, we have developed SSEhunter, a tool for the quantitative detection of alpha helices and beta sheets. Based on density skeletonization, local geometry calculations, and a template-based search, SSEhunter has been tested and validated on a variety of simulated and authentic subnanometer-resolution density maps. The result is a robust, user-friendly approach that allows users to quickly visualize, assess, and annotate intermediate-resolution density maps. Beyond secondary structure element identification, the skeletonization algorithm in SSEhunter provides secondary structure topology, which is potentially useful in leading to structural models of individual molecular components directly from the density.

    View details for DOI 10.1016/j.str.2006.11.008

    View details for PubMedID 17223528

    View details for PubMedCentralID PMC1810566

  • Single-particle electron cryomicroscopy of the ion channels in the excitation-contraction coupling junction. Methods in cell biology Serysheva, I. I., Chiu, W., Ludtke, S. J. 2007; 79: 407–35

    View details for DOI 10.1016/S0091-679X(06)79016-1

    View details for PubMedID 17327167

  • Averaging tens to hundreds of icosahedral particle images to resolve protein secondary structure elements using a Multi-Path Simulated Annealing optimization algorithm. Journal of structural biology Liu, X., Jiang, W., Jakana, J., Chiu, W. 2007; 160 (1): 11–27

    Abstract

    Accurately determining a cryoEM particle's alignment parameters is crucial to high resolution single particle 3-D reconstruction. We developed Multi-Path Simulated Annealing, a Monte-Carlo type of optimization algorithm, for globally aligning the center and orientation of a particle simultaneously. A consistency criterion was developed to ensure the alignment parameters are correct and to remove some bad particles from a large pool of images of icosahedral particles. Without using any a priori model, this procedure is able to reconstruct a structure from a random initial model. Combining the procedure above with a new empirical double threshold particle selection method, we are able to pick tens of best quality particles to reconstruct a subnanometer resolution map from scratch. Using the best 62 particles of rice dwarf virus, the reconstruction reached 9.6A resolution at which four helices of the P3A subunit of RDV are resolved. Furthermore, with the 284 best particles, the reconstruction is improved to 7.9A resolution, and 21 of 22 helices and six of seven beta sheets are resolved.

    View details for DOI 10.1016/j.jsb.2007.06.009

    View details for PubMedID 17698370

    View details for PubMedCentralID PMC2039893

  • An expanded conformation of single-ring GroEL-GroES complex encapsulates an 86 kDa substrate. Structure (London, England : 1993) Chen, D. H., Song, J. L., Chuang, D. T., Chiu, W., Ludtke, S. J. 2006; 14 (11): 1711–22

    Abstract

    Electron cryomicroscopy reveals an unprecedented conformation of the single-ring mutant of GroEL (SR398) bound to GroES in the presence of Mg-ATP. This conformation exhibits a considerable expansion of the folding cavity, with approximately 80% more volume than the X-ray structure of the equivalent cis cavity in the GroEL-GroES-(ADP)(7) complex. This expanded conformation can encapsulate an 86 kDa heterodimeric (alphabeta) assembly intermediate of mitochondrial branched-chain alpha-ketoacid dehydrogenase, the largest substrate ever observed to be cis encapsulated. The SR398-GroES-Mg-ATP complex is found to exist as a mixture of standard and expanded conformations, regardless of the absence or presence of the substrate. However, the presence of even a small substrate causes a pronounced bias toward the expanded conformation. Encapsulation of the large assembly intermediate is supported by a series of electron cryomicroscopy studies as well as the protection of both alpha and beta subunits of the substrate from tryptic digestion.

    View details for DOI 10.1016/j.str.2006.09.010

    View details for PubMedID 17098196

  • Structure of Halothiobacillus neapolitanus carboxysomes by cryo-electron tomography. Journal of molecular biology Schmid, M. F., Paredes, A. M., Khant, H. A., Soyer, F., Aldrich, H. C., Chiu, W., Shively, J. M. 2006; 364 (3): 526–35

    Abstract

    Carboxysomes are polyhedral bodies consisting of a proteinaceous shell filled with ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO). They are found in the cytoplasm of all cyanobacteria and some chemoautotrophic bacteria. Previous studies of Halothiobacillus neapolitanus and Nitrobacter agilis carboxysomes suggest that the structures are either icosahedral or dodecahedral. To determine the protein shell structure more definitively, purified H. neapolitanus carboxysomes were re-examined by cryo-electron tomography and scanning transmission electron microscopy (STEM). Due to the limited tilt angles in the electron microscope, the tomographic reconstructions are distorted. Corrections were made in the 3D orientation searching and averaging of the computationally extracted carboxysomes to minimize the missing data effects. It was found that H. neapolitanus carboxysomes vary widely in size and mass as shown by cryo-electron tomography and STEM mass measurements, respectively. We have aligned and averaged carboxysomes in several size classes from the 3D tomographic reconstruction by methods that are not model-biased. The averages reveal icosahedral symmetry of the shell, but not of the density inside it, for all the size classes.

    View details for DOI 10.1016/j.jmb.2006.09.024

    View details for PubMedID 17028023

    View details for PubMedCentralID PMC1839851

  • Structure of epsilon15 bacteriophage reveals genome organization and DNA packaging/injection apparatus. Nature Jiang, W., Chang, J., Jakana, J., Weigele, P., King, J., Chiu, W. 2006; 439 (7076): 612–16

    Abstract

    The critical viral components for packaging DNA, recognizing and binding to host cells, and injecting the condensed DNA into the host are organized at a single vertex of many icosahedral viruses. These component structures do not share icosahedral symmetry and cannot be resolved using a conventional icosahedral averaging method. Here we report the structure of the entire infectious Salmonella bacteriophage epsilon15 (ref. 1) determined from single-particle cryo-electron microscopy, without icosahedral averaging. This structure displays not only the icosahedral shell of 60 hexamers and 11 pentamers, but also the non-icosahedral components at one pentameric vertex. The densities at this vertex can be identified as the 12-subunit portal complex sandwiched between an internal cylindrical core and an external tail hub connecting to six projecting trimeric tailspikes. The viral genome is packed as coaxial coils in at least three outer layers with approximately 90 terminal nucleotides extending through the protein core and the portal complex and poised for injection. The shell protein from icosahedral reconstruction at higher resolution exhibits a similar fold to that of other double-stranded DNA viruses including herpesvirus, suggesting a common ancestor among these diverse viruses. The image reconstruction approach should be applicable to studying other biological nanomachines with components of mixed symmetries.

    View details for DOI 10.1038/nature04487

    View details for PubMedID 16452981

    View details for PubMedCentralID PMC1559657

  • Structural biology of cellular machines. Trends in cell biology Chiu, W., Baker, M. L., Almo, S. C. 2006; 16 (3): 144–50

    Abstract

    Multi-component macromolecular machines contribute to all essential biological processes, from cell motility and signal transduction to information storage and processing. Structural analysis of assemblies at atomic resolution is emerging as the field of structural cell biology. Several recent studies, including those focused on the ribosome, the acrosomal bundle and bacterial flagella, have demonstrated the ability of a hybrid approach that combines imaging, crystallography and computational tools to generate testable atomic models of fundamental biological machines. A complete understanding of cellular and systems biology will require the detailed structural understanding of hundreds of biological machines. The realization of this goal demands a concerted effort to develop and apply new strategies for the systematic identification, isolation, structural characterization and mechanistic analysis of multi-component assemblies at all resolution ranges. The establishment of a database describing the structural and dynamic properties of protein assemblies will provide novel opportunities to define the molecular and atomic mechanisms controlling overall cell physiology.

    View details for DOI 10.1016/j.tcb.2006.01.002

    View details for PubMedID 16459078

  • Cryo-EM asymmetric reconstruction of bacteriophage P22 reveals organization of its DNA packaging and infecting machinery. Structure (London, England : 1993) Chang, J., Weigele, P., King, J., Chiu, W., Jiang, W. 2006; 14 (6): 1073–82

    Abstract

    The mechanisms by which most double-stranded DNA viruses package and release their genomic DNA are not fully understood. Single particle cryo-electron microscopy and asymmetric 3D reconstruction reveal the organization of the complete bacteriophage P22 virion, including the protein channel through which DNA is first packaged and later ejected. This channel is formed by a dodecamer of portal proteins and sealed by a tail hub consisting of two stacked barrels capped by a protein needle. Six trimeric tailspikes attached around this tail hub are kinked, suggesting a functional hinge that may be used to trigger DNA release. Inside the capsid, the portal's central channel is plugged by densities interpreted as pilot/injection proteins. A short rod-like density near these proteins may be the terminal segment of the dsDNA genome. The coaxially packed DNA genome is encapsidated by the icosahedral shell. This complete structure unifies various biochemical, genetic, and crystallographic data of its components from the past several decades.

    View details for DOI 10.1016/j.str.2006.05.007

    View details for PubMedID 16730179

  • Automated segmentation of molecular subunits in electron cryomicroscopy density maps. Journal of structural biology Baker, M. L., Yu, Z., Chiu, W., Bajaj, C. 2006; 156 (3): 432–41

    Abstract

    Electron cryomicroscopy (cryoEM) is capable of imaging large macromolecular machines composed of multiple components. However, it is currently only possible to achieve moderate resolution at which it may be possible to computationally extract the individual components in the machine. In this work, we present application details of an automated method for detecting and segmenting the components of a large machine in an experimentally determined density map. This method is applicable to object with and without symmetry and takes advantage of global and local symmetry axes if present. We have applied this segmentation algorithm to several cryoEM data sets already deposited in EMDB with various complexities, symmetries and resolutions and validated the results using manually segmented density and available structures of the components in the PDB. As such, automated segmentation could become a useful tool for the analysis of the ever-increasing number of structures of macromolecular machines derived from cryoEM.

    View details for DOI 10.1016/j.jsb.2006.05.013

    View details for PubMedID 16908194

  • Cryoelectron microscopy of protein IX-modified adenoviruses suggests a new position for the C terminus of protein IX. Journal of virology Marsh, M. P., Campos, S. K., Baker, M. L., Chen, C. Y., Chiu, W., Barry, M. A. 2006; 80 (23): 11881–86

    Abstract

    Recombinant human adenovirus is a useful gene delivery vector for clinical gene therapy. Minor capsid protein IX of adenovirus has been of recent interest since multiple studies have shown that modifications can be made to its C terminus to alter viral tropism or add molecular tags and/or reporter proteins. We examined the structure of an engineered adenovirus displaying the enhanced green fluorescent protein (EGFP) fused to the C terminus of protein IX. Cryoelectron microscopy and reconstruction localized the C-terminal EGFP fusion between the H2 hexon and the H4 hexon, positioned between adjacent facets, directly above the density previously assigned as protein IIIa. The original assignment of IIIa was based largely on indirect evidence, and the data presented herein support the reassignment of the IIIa density as protein IX.

    View details for DOI 10.1128/JVI.01471-06

    View details for PubMedID 16987967

    View details for PubMedCentralID PMC1642590

  • Assessing the capabilities of a 4kx4k CCD camera for electron cryo-microscopy at 300kV. Journal of structural biology Booth, C. R., Jakana, J., Chiu, W. 2006; 156 (3): 556–63

    Abstract

    CCD cameras have numerous advantages over photographic film for detecting electrons; however the point spread function of these cameras has not been sufficient for single particle data collection to subnanometer resolution with 300kV microscopes. We have adopted spectral signal to noise ratio (SNR) as a parameter for assessing detector quality for single particle imaging. The robustness of this parameter is confirmed under a variety of experimental conditions. Using this parameter, we demonstrate that the SNR of images of either amorphous carbon film or ice embedded virus particles collected on a new commercially available 4kx4k CCD camera are slightly better than photographic film at low spatial frequency (<1/5 Nyquist frequency), and as good as photographic film out to half of the Nyquist frequency. In addition it is slightly easier to visualize ice embedded particles on this CCD camera than on photographic film. Based on this analysis it is realistic to collect images containing subnanometer resolution data (6-9A) using this CCD camera at an effective magnification of approximately 112000x on a 300kV electron microscope.

    View details for DOI 10.1016/j.jsb.2006.08.019

    View details for PubMedID 17067819

  • Ab initio modeling of the herpesvirus VP26 core domain assessed by CryoEM density. PLoS computational biology Baker, M. L., Jiang, W., Wedemeyer, W. J., Rixon, F. J., Baker, D., Chiu, W. 2006; 2 (10): e146

    Abstract

    Efforts in structural biology have targeted the systematic determination of all protein structures through experimental determination or modeling. In recent years, 3-D electron cryomicroscopy (cryoEM) has assumed an increasingly important role in determining the structures of these large macromolecular assemblies to intermediate resolutions (6-10 A). While these structures provide a snapshot of the assembly and its components in well-defined functional states, the resolution limits the ability to build accurate structural models. In contrast, sequence-based modeling techniques are capable of producing relatively robust structural models for isolated proteins or domains. In this work, we developed and applied a hybrid modeling approach, utilizing cryoEM density and ab initio modeling to produce a structural model for the core domain of a herpesvirus structural protein, VP26. Specifically, this method, first tested on simulated data, utilizes the cryoEM density map as a geometrical constraint in identifying the most native-like models from a gallery of models generated by ab initio modeling. The resulting model for the core domain of VP26, based on the 8.5-A resolution herpes simplex virus type 1 (HSV-1) capsid cryoEM structure and mutational data, exhibited a novel fold. Additionally, the core domain of VP26 appeared to have a complementary interface to the known upper-domain structure of VP5, its cognate binding partner. While this new model provides for a better understanding of the assembly and interactions of VP26 in HSV-1, the approach itself may have broader applications in modeling the components of large macromolecular assemblies.

    View details for DOI 10.1371/journal.pcbi.0020146

    View details for PubMedID 17069457

    View details for PubMedCentralID PMC1626159

  • Common ancestry of herpesviruses and tailed DNA bacteriophages. Journal of virology Baker, M. L., Jiang, W., Rixon, F. J., Chiu, W. 2005; 79 (23): 14967–70

    Abstract

    Comparative analysis of capsid protein structures in the eukaryote-infecting herpesviruses (Herpesviridae) and the prokaryote-infecting tailed DNA bacteriophages (Caudovirales) revealed a characteristic fold that is restricted to these two virus lineages and is indicative of common ancestry. This fold not only serves as a major architectural element in capsid stability but also enables the conformational flexibility observed during viral assembly and maturation. On the basis of this and other emerging relationships, it seems increasingly likely that the very diverse collection of extant viruses may have arisen from a relatively small number of primordial progenitors.

    View details for DOI 10.1128/JVI.79.23.14967-14970.2005

    View details for PubMedID 16282496

    View details for PubMedCentralID PMC1287596

  • Structure of Ca2+ release channel at 14 A resolution. Journal of molecular biology Serysheva, I. I., Hamilton, S. L., Chiu, W., Ludtke, S. J. 2005; 345 (3): 427–31

    Abstract

    The 14 A resolution structure of the 2.3 MDa Ca2+ release channel (also known as RyR1) was determined by electron cryomicroscopy and single particle reconstruction. This structure was produced using collected data used for our previous published structures at 22-30 A resolution, but now taking advantage of recent algorithmic improvements in the EMAN software suite. This improved map clearly exhibits more structural detail and allows better defined docking of computationally predicted structural domain folds. Using sequence-based fold recognition, the N-terminal region of RyR1, residues 216-572, was predicted to have significant structural similarity with the IP3-binding core region of the type 1 IP3R. This putative structure was computationally localized to the clamp-shaped region of RyR1, which has been implicated to have a regulatory role in the channel activity.

    View details for DOI 10.1016/j.jmb.2004.10.073

    View details for PubMedID 15581887

    View details for PubMedCentralID PMC2978512

  • Electron cryomicroscopy of biological machines at subnanometer resolution. Structure (London, England : 1993) Chiu, W., Baker, M. L., Jiang, W., Dougherty, M., Schmid, M. F. 2005; 13 (3): 363–72

    Abstract

    Advances in electron cryomicroscopy (cryo-EM) have made possible the structural determination of large biological machines in the resolution range of 6-9 angstroms. Rice dwarf virus and the acrosomal bundle represent two distinct types of machines amenable to cryo-EM investigations at subnanometer resolutions. However, calculating the density map is only the first step, and much analysis remains to extract structural insights and the mechanism of action in these machines. This paper will review the computational and visualization methodologies necessary for analysis (structure mining) of the computed cryo-EM maps of these machines. These steps include component segmentation, averaging based on local symmetry among components, density connectivity trace, incorporation of bioinformatics analysis, and fitting of high-resolution component data, if available. The consequences of these analyses can not only identify accurately some of the secondary structure elements of the molecular components in machines but also suggest structural mechanisms related to their biological functions.

    View details for DOI 10.1016/j.str.2004.12.016

    View details for PubMedID 15766537

  • The pore structure of the closed RyR1 channel. Structure (London, England : 1993) Ludtke, S. J., Serysheva, I. I., Hamilton, S. L., Chiu, W. 2005; 13 (8): 1203–11

    Abstract

    Using single particle electron cryomicroscopy, several helices in the membrane-spanning region of RyR1, including an inner transmembrane helix, a short pore helix, and a helix parallel to the membrane on the cytoplasmic side, have been clearly resolved. Our model places a highly conserved glycine (G4934) at the hinge position of the bent inner helix and two rings of negative charges at the luminal and cytoplasmic mouths of the pore. The kinked inner helix closely resembles the inner helix of the open MthK channel, suggesting that kinking alone does not open RyR1, as proposed for K+ channels.

    View details for DOI 10.1016/j.str.2005.06.005

    View details for PubMedID 16084392

    View details for PubMedCentralID PMC2983469

  • A digital atlas to characterize the mouse brain transcriptome. PLoS computational biology Carson, J. P., Ju, T., Lu, H. C., Thaller, C., Xu, M., Pallas, S. L., Crair, M. C., Warren, J., Chiu, W., Eichele, G. 2005; 1 (4): e41

    Abstract

    Massive amounts of data are being generated in an effort to represent for the brain the expression of all genes at cellular resolution. Critical to exploiting this effort is the ability to place these data into a common frame of reference. Here we have developed a computational method for annotating gene expression patterns in the context of a digital atlas to facilitate custom user queries and comparisons of this type of data. This procedure has been applied to 200 genes in the postnatal mouse brain. As an illustration of utility, we identify candidate genes that may be related to Parkinson disease by using the expression of a dopamine transporter in the substantia nigra as a search query pattern. In addition, we discover that transcription factor Rorb is down-regulated in the barrelless mutant relative to control mice by quantitative comparison of expression patterns in layer IV somatosensory cortex. The semi-automated annotation method developed here is applicable to a broad spectrum of complex tissues and data modalities.

    View details for DOI 10.1371/journal.pcbi.0010041

    View details for PubMedID 16184189

    View details for PubMedCentralID PMC1215388

  • Conformational changes in Sindbis virions resulting from exposure to low pH and interactions with cells suggest that cell penetration may occur at the cell surface in the absence of membrane fusion VIROLOGY Paredes, A. M., Ferreira, D., Horton, M., Saad, A., Tsuruta, H., Johnston, R., Klimstra, W., Ryman, K., Hernandez, R., Chiu, W., Brown, D. T. 2004; 324 (2): 373-386

    Abstract

    Alphaviruses have the ability to induce cell-cell fusion after exposure to acid pH. This observation has served as an article of proof that these membrane-containing viruses infect cells by fusion of the virus membrane with a host cell membrane upon exposure to acid pH after incorporation into a cell endosome. We have investigated the requirements for the induction of virus-mediated, low pH-induced cell-cell fusion and cell-virus fusion. We have correlated the pH requirements for this process to structural changes they produce in the virus by electron cryo-microscopy. We found that exposure to acid pH was required to establish conditions for membrane fusion but that membrane fusion did not occur until return to neutral pH. Electron cryo-microscopy revealed dramatic changes in the structure of the virion as it was moved to acid pH and then returned to neutral pH. None of these treatments resulted in the disassembly of the virus protein icosahedral shell that is a requisite for the process of virus membrane-cell membrane fusion. The appearance of a prominent protruding structure upon exposure to acid pH and its disappearance upon return to neutral pH suggested that the production of a "pore"-like structure at the fivefold axis may facilitate cell penetration as has been proposed for polio (J. Virol. 74 (2000) 1342) and human rhino virus (Mol. Cell 10 (2002) 317). This transient structural change also provided an explanation for how membrane fusion occurs after return to neutral pH. Examination of virus-cell complexes at neutral pH supported the contention that infection occurs at the cell surface at neutral pH by the production of a virus structure that breaches the plasma membrane bilayer. These data suggest an alternative route of infection for Sindbis virus that occurs by a process that does not involve membrane fusion and does not require disassembly of the virus protein shell.

    View details for DOI 10.1016/j.virus.2004.03.046

    View details for Web of Science ID 000222376800012

    View details for PubMedID 15207623

  • Structure of the acrosomal bundle. Nature Schmid, M. F., Sherman, M. B., Matsudaira, P., Chiu, W. 2004; 431 (7004): 104–7

    Abstract

    In the unactivated Limulus sperm, a 60- micro m-long bundle of actin filaments crosslinked by the protein scruin is bent and twisted into a coil around the base of the nucleus. At fertilization, the bundle uncoils and fully extends in five seconds to support a finger of membrane known as the acrosomal process. This biological spring is powered by stored elastic energy and does not require the action of motor proteins or actin polymerization. In a 9.5-A electron cryomicroscopic structure of the extended bundle, we show that twist, tilt and rotation of actin-scruin subunits deviate widely from a 'standard' F-actin filament. This variability in structural organization allows filaments to pack into a highly ordered and rigid bundle in the extended state and suggests a mechanism for storing and releasing energy between coiled and extended states without disassembly.

    View details for DOI 10.1038/nature02881

    View details for PubMedID 15343340

  • Experimental verification of conformational variation of human fatty acid synthase as predicted by normal mode analysis. Structure (London, England : 1993) Brink, J., Ludtke, S. J., Kong, Y., Wakil, S. J., Ma, J., Chiu, W. 2004; 12 (2): 185–91

    Abstract

    Fatty acid synthase (FAS) is a 550 kDa homodimeric enzyme with multiple functional and structural domains. Normal mode analysis of a previously determined 19 A structure of FAS suggested that this enzyme might assume different conformational states with several distinct hinge movements. We have used a simultaneous multiple-model refinement method to search for the presence of the structural conformers from the electron images of FAS. We have demonstrated that the resulting models observed in the electron images are consistent with the predicted conformational changes. This technique demonstrates the potential of the combination of normal mode analysis with multiple model refinement to elucidate the multiple conformations of flexible proteins. Since each of these structures is based on a more homogeneous particle set, this technique has the potential, provided that sufficient references are used, to improve the resolution of the final reconstructions of single particles from electron cryomicroscopy.

    View details for DOI 10.1016/j.str.2004.01.015

    View details for PubMedID 14962379

  • A 9 angstroms single particle reconstruction from CCD captured images on a 200 kV electron cryomicroscope. Journal of structural biology Booth, C. R., Jiang, W., Baker, M. L., Zhou, Z. H., Ludtke, S. J., Chiu, W. 2004; 147 (2): 116–27

    Abstract

    Sub-nanometer resolution structure determination is becoming a common practice in electron cryomicroscopy of macromolecular assemblies. The data for these studies have until now been collected on photographic film. Using cytoplasmic polyhedrosis virus (CPV), a previously determined structure, as a test specimen, we show the feasibility of obtaining a 9 angstroms structure from images acquired from a 4 k x 4 k Gatan CCD on a 200 kV electron cryomicroscope. The match of the alpha-helices in the protein components of the CPV with the previous structure of the same virus validates the suitability of this type of camera as the recording media targeted for single particle reconstructions at sub-nanometer resolution.

    View details for DOI 10.1016/j.jsb.2004.02.004

    View details for PubMedID 15193640

  • Seeing GroEL at 6 A resolution by single particle electron cryomicroscopy. Structure (London, England : 1993) Ludtke, S. J., Chen, D. H., Song, J. L., Chuang, D. T., Chiu, W. 2004; 12 (7): 1129–36

    Abstract

    We present a reconstruction of native GroEL by electron cryomicroscopy (cryo-EM) and single particle analysis at 6 A resolution. alpha helices are clearly visible and beta sheet density is also visible at this resolution. While the overall conformation of this structure is quite consistent with the published X-ray data, a measurable shift in the positions of three alpha helices in the intermediate domain is observed, not consistent with any of the 7 monomeric structures in the Protein Data Bank model (1OEL). In addition, there is evidence for slight rearrangement or flexibility in parts of the apical domain. The 6 A resolution cryo-EM GroEL structure clearly demonstrates the veracity and expanding scope of cryo-EM and the single particle reconstruction technique for macromolecular machines.

    View details for DOI 10.1016/j.str.2004.05.006

    View details for PubMedID 15242589

  • Structure of the bifunctional and Golgi-associated formiminotransferase cyclodeaminase octamer. The EMBO journal Mao, Y., Vyas, N. K., Vyas, M. N., Chen, D. H., Ludtke, S. J., Chiu, W., Quiocho, F. A. 2004; 23 (15): 2963–71

    Abstract

    Mammalian formiminotransferase cyclodeaminase (FTCD), a 0.5 million Dalton homo-octameric enzyme, plays important roles in coupling histidine catabolism with folate metabolism and integrating the Golgi complex with the vimentin intermediate filament cytoskeleton. It is also linked to two human diseases, autoimmune hepatitis and glutamate formiminotransferase deficiency. Determination of the FTCD structure by X-ray crystallography and electron cryomicroscopy revealed that the eight subunits, each composed of distinct FT and CD domains, are arranged like a square doughnut. A key finding indicates that coupling of three subunits governs the octamer-dependent sequential enzyme activities, including channeling of intermediate and conformational change. The structure further shed light on the molecular nature of two strong antigenic determinants of FTCD recognized by autoantibodies from patients with autoimmune hepatitis and on the binding of thin vimentin filaments to the FTCD octamer.

    View details for DOI 10.1038/sj.emboj.7600327

    View details for PubMedID 15272307

    View details for PubMedCentralID PMC514939

  • Electron beam coater for reduction of charging in ice-embedded biological specimens using Ti(88)Si(12) alloy. Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada Sherman, M. B., Chiu, W. 2003; 9 (6): 566–73

    Abstract

    Biological macromolecules embedded in vitreous ice are known to suffer from charging while being imaged in an electron transmission cryomicroscope. We developed an electron beam coater that deposits conductive films onto the surface of frozen-hydrated specimens. The conductive films help to dissipate charge during electron irradiation of poorly conductive ice-embedded biological samples. We observed significant reduction in charging of ice-embedded catalase crystals suspended over holes in a holey carbon film after coating them with a 30-A-thick layer of an amorphous alloy, Ti(88)Si(12). Images of the crystals after coating showed diffraction spots of up to 3 A resolution.

    View details for DOI 10.1017/S1431927603030435

    View details for PubMedID 14750991

  • Structure of isolated nucleocapsids from venezuelan equine encephalitis virus and implications for assembly and disassembly of enveloped virus. Journal of virology Paredes, A., Alwell-Warda, K., Weaver, S. C., Chiu, W., Watowich, S. J. 2003; 77 (1): 659–64

    Abstract

    Venezuelan equine encephalitis virus (VEEV) is an important human and equine pathogen in the Americas, with widespread reoccurring epidemics extending from South America to the southern United States. Most troubling, VEEV has been made into a weapon by several countries and is currently restricted by the Centers for Disease Control and Prevention as a potential biological warfare and terrorism agent. To facilitate the development of antiviral compounds, the structure of the nucleocapsid isolated from VEEV has been determined by electron cryomicroscopy and image reconstruction and represents the first three-dimensional structure of a nucleocapsid isolated from a single-stranded enveloped RNA virus. The isolated VEEV nucleocapsid undergoes significant reorganization relative to its structure within VEEV. However, the isolated nucleocapsid clearly exhibits T=4 icosahedral symmetry, and its characteristic nucleocapsid hexons and pentons are preserved. The diameter of the isolated nucleocapsid is approximately 11.5% larger than that of the nucleocapsid within VEEV, with radial expansion being greatest near the hexons. Significantly, this is the first direct structural evidence showing that a simple enveloped virus undergoes large conformational changes during maturation, suggesting that the lipid bilayer and the transmembrane proteins of simple enveloped viruses provide the energy necessary to reorganize the nucleocapsid during maturation.

    View details for DOI 10.1128/jvi.77.1.659-664.2003

    View details for PubMedID 12477868

    View details for PubMedCentralID PMC140571

  • Coat protein fold and maturation transition of bacteriophage P22 seen at subnanometer resolutions. Nature structural biology Jiang, W., Li, Z., Zhang, Z., Baker, M. L., Prevelige, P. E., Chiu, W. 2003; 10 (2): 131–35

    Abstract

    Bacteriophage P22 is a prototypical biological machine used for studying protein complex assembly and capsid maturation. Using cryo-EM, we solved the structures of P22 before and after the capsid maturation at 8.5 A and 9.5 A resolutions, respectively. These structures allowed visualization of alpha-helices and beta-sheets from which the capsid protein fold is derived. The capsid fold is similar to that of the coat protein of HK97 bacteriophage. The cryo-EM shows that a large conformational change of the P22 capsid during maturation transition involves not only the domain movement of individual subunits, but also refolding of the capsid protein.

    View details for DOI 10.1038/nsb891

    View details for PubMedID 12536205

  • Structure of the herpesvirus major capsid protein. The EMBO journal Bowman, B. R., Baker, M. L., Rixon, F. J., Chiu, W., Quiocho, F. A. 2003; 22 (4): 757–65

    Abstract

    Herpes simplex virus-1 (HSV-1) virions are large, complex enveloped particles containing a proteinaceous tegument layer connected to an icosahedral capsid. The major capsid protein, VP5 (149 kDa), makes up both types of capsomere, pentons and hexons. Limited trypsin digestion of VP5 identified a single stable 65 kDa fragment which represents a proposed protein folding nucleus. We report the 2.9 A crystal structure of this fragment and its modeling into an 8.5 A resolution electron cryomicroscopy map of the HSV-1 capsid. The structure, the first for any capsid protein from Herpesviridae, revealed a novel fold, placing herpesviruses outside any of the structurally linked viral groupings. Alterations in the geometrical arrangements of the VP5 subunits in the capsomeres exposes different residues, resulting in the differential association of the tegument and VP26 with the pentons and hexons, respectively. The rearrangements of VP5 subunits required to form both pentavalent and hexavalent capsomeres result in structures that exhibit very different electrostatic properties. These differences may mediate the binding and release of other structural proteins during capsid maturation.

    View details for DOI 10.1093/emboj/cdg086

    View details for PubMedID 12574112

    View details for PubMedCentralID PMC145446

  • Structure of the type 1 inositol 1,4,5-trisphosphate receptor revealed by electron cryomicroscopy. The Journal of biological chemistry Serysheva, I. I., Bare, D. J., Ludtke, S. J., Kettlun, C. S., Chiu, W., Mignery, G. A. 2003; 278 (24): 21319–22

    Abstract

    The three-dimensional structure of the type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1) has been determined by electron cryomicroscopy and single-particle reconstruction. The receptor was immunoaffinity-purified and formed functional InsP3- and heparin-sensitive channels with a unitary conductance similar to native InsP3Rs. The channel structure exhibits the expected 4-fold symmetry and comprises two morphologically distinct regions: a large pinwheel and a smaller square. The pinwheel region has four radial curved spokes interconnected by a central core. The InsP3-binding core domain has been localized within each spoke of the pinwheel region by fitting its x-ray structure into our reconstruction. A structural mapping of the amino acid sequences to several functional domains is deduced within the structure of the InsP3R1 tetramer.

    View details for DOI 10.1074/jbc.C300148200

    View details for PubMedID 12714606

  • Architecture of the herpes simplex virus major capsid protein derived from structural bioinformatics. Journal of molecular biology Baker, M. L., Jiang, W., Bowman, B. R., Zhou, Z. H., Quiocho, F. A., Rixon, F. J., Chiu, W. 2003; 331 (2): 447–56

    Abstract

    The dispositions of 39 alpha helices of greater than 2.5 turns and four beta sheets in the major capsid protein (VP5, 149 kDa) of herpes simplex virus type 1 were identified by computational and visualization analysis from the 8.5A electron cryomicroscopy structure of the whole capsid. The assignment of helices in the VP5 upper domain was validated by comparison with the recently determined crystal structure of this region. Analysis of the spatial arrangement of helices in the middle domain of VP5 revealed that the organization of a tightly associated bundle of ten helices closely resembled that of a domain fold found in the annexin family of proteins. Structure-based sequence searches suggested that sequences in both the N and C-terminal portions of the VP5 sequence contribute to this domain. The long helices seen in the floor domain of VP5 form an interconnected network within and across capsomeres. The combined structural and sequence-based informatics has led to an architectural model of VP5. This model placed in the context of the capsid provides insights into the strategies used to achieve viral capsid stability.

    View details for DOI 10.1016/s0022-2836(03)00696-x

    View details for PubMedID 12888351

  • The structure of ClpB: a molecular chaperone that rescues proteins from an aggregated state. Cell Lee, S., Sowa, M. E., Watanabe, Y. H., Sigler, P. B., Chiu, W., Yoshida, M., Tsai, F. T. 2003; 115 (2): 229–40

    Abstract

    Molecular chaperones assist protein folding by facilitating their "forward" folding and preventing aggregation. However, once aggregates have formed, these chaperones cannot facilitate protein disaggregation. Bacterial ClpB and its eukaryotic homolog Hsp104 are essential proteins of the heat-shock response, which have the remarkable capacity to rescue stress-damaged proteins from an aggregated state. We have determined the structure of Thermus thermophilus ClpB (TClpB) using a combination of X-ray crystallography and cryo-electron microscopy (cryo-EM). Our single-particle reconstruction shows that TClpB forms a two-tiered hexameric ring. The ClpB/Hsp104-linker consists of an 85 A long and mobile coiled coil that is located on the outside of the hexamer. Our mutagenesis and biochemical data show that both the relative position and motion of this coiled coil are critical for chaperone function. Taken together, we propose a mechanism by which an ATP-driven conformational change is coupled to a large coiled-coil motion, which is indispensable for protein disaggregation.

    View details for DOI 10.1016/s0092-8674(03)00807-9

    View details for PubMedID 14567920

  • Structure of triglyceride-rich human low-density lipoproteins according to cryoelectron microscopy. Biochemistry Sherman, M. B., Orlova, E. V., Decker, G. L., Chiu, W., Pownall, H. J. 2003; 42 (50): 14988–93

    Abstract

    Low-density lipoprotein (LDL) particles from normolipidemic individuals contain a cholesteryl ester-rich core that undergoes a thermal transition from a liquid crystalline to an isotropic liquid phase between 20 and 35 degrees C. LDL from hypertriglyceridemic patients or prepared in vitro by the exchange of very low-density lipoprotein for LDL cholesteryl esters is triglyceride-rich, does not have a thermal transition above 0 degrees C, and exhibits impaired binding to the LDL receptor on normal human skin fibroblasts. Cryoelectron microscopy of LDL quick-frozen from 10 (core-frozen) and 40 degrees C (core-melted) revealed ellipsoidal particles with internal striations and round particles devoid of striations, respectively. Cryoelectron microscopy of triglyceride-rich LDL prepared in vitro revealed particles similar to the core-melted normolipidemic LDL, i.e., round particles without striations. These data suggest that the LDL core in the liquid crystalline phase is characterized by the appearance of striations, whereas LDL with a core that is an isotropic liquid lacks striations. It is suggested that freezing the LDL core into a liquid crystalline phase imposes structural constraints that force LDL from a sphere without partitions to an ellipsoid with partitions. We further suggest that the striation-defined lamellae are a structural feature of a liquid crystalline neutral lipid core that is a determinant of normal binding to the LDL receptor and that conversion of the neutral lipid core of LDL to the isotropic liquid phase via an increase in the temperature or via the addition of triglyceride partially ablates the receptor binding determinants on the LDL surface. This effect is likely achieved through changes in the conformation of apo-B-100. These data suggest that the physical state of the LDL core determines particle shape, surface structure, and metabolic fate.

    View details for DOI 10.1021/bi0354738

    View details for PubMedID 14674775

  • Object oriented database and electronic notebook for transmission electron microscopy. Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada Ludtke, S. J., Nason, L., Tu, H., Peng, L., Chiu, W. 2003; 9 (6): 556–65

    Abstract

    As high-resolution biological transmission electron microscopy (TEM) has increased in popularity over recent years, the volume of data and number of projects underway has risen dramatically. A robust tool for effective data management is essential to efficiently process large data sets and extract maximum information from the available data. We present the Electron Microscopy Electronic Notebook (EMEN), a portable, object-oriented, web-based tool for TEM data archival and project management. EMEN has several unique features. First, the database is logically organized and annotated so multiple collaborators at different geographical locations can easily access and interpret the data without assistance. Second, the database was designed to provide flexibility to the user, so it can be used much as a lab notebook would be, while maintaining a structure suitable for data mining and direct interaction with data-processing software. Finally, as an object-oriented database, the database structure is dynamic and can be easily extended to incorporate information not defined in the original database specification.

    View details for DOI 10.1017/S1431927603030575

    View details for PubMedID 14750990

  • Electron cryo-microscopy of VAT, the archaeal p97/CDC48 homologue from Thermoplasma acidophilum JOURNAL OF MOLECULAR BIOLOGY Rockel, B., Jakana, J., Chiu, W., Baumeister, W. 2002; 317 (5): 673-681

    Abstract

    VAT (valosine containing protein-like ATPase from Thermoplasma acidophilum), an archaeal member of the AAA-family (ATPases associated with a variety of cellular activities) that possesses foldase as well as unfoldase-activity, forms homo-hexameric rings like its eukaryotic homologues p97 and CDC48. The VAT-monomer exhibits the tripartite domain architecture typical for type II AAA-ATPases: N-D1-D2, whereby N is the substrate binding N-terminal domain preceding domains D1 and D2, both containing AAA-modules. Recent 3-D reconstructions of VAT and p97 as obtained by electron microscopy suffer from weakly represented N-domains, probably a consequence of their flexible linkage to the hexameric core. Here we used electron cryo-microscopy and 3-D reconstruction of single particles in order to generate a 3-D model of VAT at 2.3 nm resolution. The hexameric core of the VAT-complex (diameter 13.2 nm, height 8.4 nm) encloses a central cavity and the substrate-binding N-domains are clearly arranged in the upper periphery. Comparison with the p97 3-D reconstruction and the recently determined crystal structure of p97-N-D1 suggests a tail-to-tail arrangement of D1 and D2 in VAT.

    View details for DOI 10.1006/jmbi.2002.5448

    View details for Web of Science ID 000175202300003

    View details for PubMedID 11955016

  • The skeletal muscle Ca2+ release channel has an oxidoreductase-like domain. Proceedings of the National Academy of Sciences of the United States of America Baker, M. L., Serysheva, I. I., Sencer, S., Wu, Y., Ludtke, S. J., Jiang, W., Hamilton, S. L., Chiu, W. 2002; 99 (19): 12155–60

    Abstract

    We used a combination of bioinformatics, electron cryomicroscopy, and biochemical techniques to identify an oxidoreductase-like domain in the skeletal muscle Ca2+ release channel protein (RyR1). The initial prediction was derived from sequence-based fold recognition for the N-terminal region (41-420) of RyR1. The putative domain was computationally localized to the clamp domain in the cytoplasmic region of a 22A structure of RyR1. This localization was subsequently confirmed by difference imaging with a sequence specific antibody. Consistent with the prediction of an oxidoreductase domain, RyR1 binds [3H]NAD+, supporting a model in which RyR1 has a oxidoreductase-like domain that could function as a type of redox sensor.

    View details for DOI 10.1073/pnas.182058899

    View details for PubMedID 12218169

    View details for PubMedCentralID PMC129414

  • Quaternary structure of human fatty acid synthase by electron cryomicroscopy. Proceedings of the National Academy of Sciences of the United States of America Brink, J., Ludtke, S. J., Yang, C. Y., Gu, Z. W., Wakil, S. J., Chiu, W. 2002; 99 (1): 138–43

    Abstract

    We present the first three-dimensional reconstruction of human fatty acid synthase obtained by electron cryomicroscopy and single-particle image processing. The structure shows that the synthase is composed of two monomers, arranged in an antiparallel orientation, which is consistent with biochemical data. The monomers are connected to each other at their middle by a bridge of density, a site proposed to be the combination of the interdomain regions of the two monomers. Each monomer subunit appears to be subdivided into three structural domains. With this reconstruction of the synthase, we propose a location for the enzyme's two fatty acid synthesis sites.

    View details for DOI 10.1073/pnas.012589499

    View details for PubMedID 11756679

    View details for PubMedCentralID PMC117528

  • Bilamellar cationic liposomes protect adenovectors from preexisting humoral immune responses. Molecular therapy : the journal of the American Society of Gene Therapy Yotnda, P., Chen, D. H., Chiu, W., Piedra, P. A., Davis, A., Templeton, N. S., Brenner, M. K. 2002; 5 (3): 233–41

    Abstract

    Adenoviral vectors have been widely used for gene therapy, but they are limited both by the presence of a humoral immune response that dramatically decreases the level of transduction after reinjection and by their requirement for target cells to express appropriate receptors such as Coxsackie adenovirus receptor (CAR). To overcome both limits, we encapsulated adenovectors using bilamellar DOTAP:chol liposomes. Electron micrography (EM) showed that these liposomes efficiently encapsulated the vectors, allowing CAR-independent adenovector transduction of otherwise resistant cells. DOTAP:chol-encapsulated adenovectors encoding LacZ or alpha(1)-antitrypsin inhibitor (AAT) were also functionally resistant ex vivo and in vivo to the neutralizing effects of human anti-adenoviral antibodies, unlike other liposomal systems. Hence, bilamellar DOTAP:chol liposomes may be useful for applications using adenovectors in which the target cells lack adenoviral receptors or in which the recipient already has or develops a neutralizing antibody response that would otherwise inactivate readministered vector.

    View details for DOI 10.1006/mthe.2002.0545

    View details for PubMedID 11863412

  • High resolution structural studies of complex icosahedral viruses: a brief overview. Virus research Chiu, W., Rixon, F. J. 2002; 82 (1-2): 9–17

    Abstract

    Structural descriptions of viral particles are key to our understanding of their assembly mechanisms and properties. We will describe the application of X-ray crystallography and electron cryomicroscopy to the structural determination of the bluetongue virus core and the herpesvirus capsid. These represent the highest resolution structural studies carried out by these techniques on such complex and large icosahedral virus particles. The bluetongue virus core consists of two layers of distinct proteins with different protein packing symmetries, while the herpes virus capsid is made up of four types of proteins with 3.3 MDa per asymmetric unit. The structural results reveal that each of these proteins has distinct folds and they are packed uniquely to form stable particles.

    View details for DOI 10.1016/s0168-1702(01)00381-1

    View details for PubMedID 11885957

  • Deriving folds of macromolecular complexes through electron cryomicroscopy and bioinformatics approaches. Current opinion in structural biology Chiu, W., Baker, M. L., Jiang, W., Zhou, Z. H. 2002; 12 (2): 263–69

    Abstract

    Intermediate-resolution (7-9A) structures of large macromolecular complexes can be obtained by electron cryomicroscopy. This structural information, combined with bioinformatics data for the individual protein components or domains, can lead to a fold model for the entire complex. Such approaches have been demonstrated with the 6.8 A structure of the rice dwarf virus to derive models for the major capsid shell proteins.

    View details for DOI 10.1016/s0959-440x(02)00319-6

    View details for PubMedID 11959506

  • Fourier amplitude decay of electron cryomicroscopic images of single particles and effects on structure determination JOURNAL OF STRUCTURAL BIOLOGY Saad, A., Ludtke, S. J., Jakana, J., Rixon, F. J., Tsuruta, H., Chiu, W. 2001; 133 (1): 32-42

    Abstract

    Several factors, including spatial and temporal coherence of the electron microscope, specimen movement, recording medium, and scanner optics, contribute to the decay of the measured Fourier amplitude in electron image intensities. We approximate the combination of these factors as a single Gaussian envelope function, the width of which is described by a single experimental B-factor. We present an improved method for estimating this B-factor from individual micrographs by combining the use of X-ray solution scattering and numerical fitting to the average power spectrum of particle images. A statistical estimation from over 200 micrographs of herpes simplex virus type-1 capsids was used to estimate the spread in the experimental B-factor of the data set. The B-factor is experimentally shown to be dependent on the objective lens defocus setting of the microscope. The average B-factor, the X-ray scattering intensity of the specimen, and the number of particles required to determine the structure at a lower resolution can be used to estimate the minimum fold increase in the number of particles that would be required to extend a single particle reconstruction to a specified higher resolution. We conclude that microscope and imaging improvements to reduce the experimental B-factor will be critical for obtaining an atomic resolution structure.

    View details for DOI 10.1006/jsbi.2001.4330

    View details for Web of Science ID 000169093800004

    View details for PubMedID 11356062

  • Web-based Simulation for Contrast Transfer Function and Envelope Functions. Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada Jiang, W., Chiu, W. 2001; 7 (4): 329–34

    Abstract

    We have implemented a Web-based simulation program for the contrast transfer function and envelope functions in Java and JavaScript. The simulation provides interactive controls of all the parameters in those functions. In addition to the predefined functions, users can easily define new functions that use any of the parameters in the preset functions. The most useful feature of this new simulation program is the convenient, universal accessibility through Web browsers on any computer platform that supports Java, such as Netscape and Internet Explorer.

    View details for DOI 10.1017.S1431927601010315

    View details for PubMedID 12597807

  • CTF corrected stucture of alpha-crystallin B by electron cyro-microscopy. Baldwin, P. R., Ludtke, S. J., Serysheva, Cobb, B. A., Quiocho, F., Petrash, M., Tsuruta, H., Chiu, W. BIOPHYSICAL SOCIETY. 2000: 8A
  • Scaling structure factor amplitudes in electron cryomicroscopy using X-ray solution scattering JOURNAL OF STRUCTURAL BIOLOGY Schmid, M. F., Sherman, M. B., Matsudaira, P., Tsuruta, H., Chiu, W. 1999; 128 (1): 51-57

    Abstract

    The structure factors derived from electron cryomicroscopic images are modified by the contrast transfer function of the microscope's objective lens and other influences. The phases of the structure factors can be corrected in a straightforward way when the positions of the contrast transfer function rings are determined. However, corrected amplitudes are also essential to yield an accurate distribution of mass in the reconstruction. The correct scale factors for the amplitudes are difficult to evaluate for data that are merged from many different micrographs. We opt to use X-ray solution scattering intensity from a concentrated suspension of the specimen to correct the amplitudes of the spherically averaged structure factors. When this approach is applied to the three-dimensional image data of ice-embedded acrosomal bundles, the core of a filament in a three-dimensional reconstruction of the acrosomal bundle becomes denser and matches more closely the outer density ascribed to scruin.

    View details for Web of Science ID 000084451600008

    View details for PubMedID 10600558

  • Solution X-ray scattering-based estimation of electron cryomicroscopy imaging parameters for reconstruction of virus particles BIOPHYSICAL JOURNAL Thuman-Commike, P. A., Tsuruta, H., Greene, B., Prevelige, P. E., KING, J., Chiu, W. 1999; 76 (4): 2249-2261

    Abstract

    Structure factor amplitudes and phases can be computed directly from electron cryomicroscopy images. Inherent aberrations of the electromagnetic lenses and other instrumental factors affect the structure factors, however, resulting in decreased accuracy in the determined three-dimensional reconstruction. In contrast, solution x-ray scattering provides absolute and accurate measurement of spherically averaged structure factor amplitudes of particles in solution but does not provide information on the phases. In the present study, we explore the merits of using solution x-ray scattering data to estimate the imaging parameters necessary to make corrections to the structure factor amplitudes derived from electron cryomicroscopic images of icosahedral virus particles. Using 400-kV spot-scan images of the bacteriophage P22 procapsid, we have calculated an amplitude contrast of 8.0 +/- 5.2%. The amplitude decay parameter has been estimated to be 523 +/- 188 A2 with image noise compensation and 44 +/- 66 A2 without it. These results can also be used to estimate the minimum number of virus particles needed for reconstruction at different resolutions.

    View details for Web of Science ID 000079643400049

    View details for PubMedID 10096920

    View details for PubMedCentralID PMC1300198

  • COLD STAGE DESIGN FOR HIGH-RESOLUTION ELECTRON-MICROSCOPY OF BIOLOGICAL-MATERIALS ELECTRON MICROSCOPY REVIEWS Downing, K. H., Chiu, W. 1990; 3 (2): 213-226

    Abstract

    Both the number and range of applications of cryotechniques in transmission electron microscopy are increasing rapidly. In some cases, most notably the determination of protein structure by electron crystallography, progress has been limited by the performance of commercially available cryo stages. We review the design and performance criteria for stages which will be necessary for wide applicability in high resolution studies of biological specimens. The important criteria include an operating temperature below -140 degrees C with a low rate of contamination of the specimen, ability to tilt to 60 degrees, and perhaps most important, good resolution as judged by an effective modulation transfer function of 0.8 at 0.35 nm. Most applications also require an effective cryotransfer system. Up until now, most work in high resolution electron crystallography has been accomplished with laboratory-built stages which meet some, but not all, of these criteria. The availability of cold stages which fully meet criteria will allow the rapid expansion of high resolution studies by electron microscopy in structural biology.

    View details for Web of Science ID A1990EG78800002

    View details for PubMedID 2103342

  • ALIGNMENT AND MERGING OF ELECTRON-MICROSCOPE IMAGES OF FROZEN HYDRATED CRYSTALS OF THE T4 DNA HELIX DESTABILIZING PROTEIN GP32-STAR-I BIOPHYSICAL JOURNAL Grant, R. A., Schmid, M. F., Chiu, W., DEATHERAGE, J. F., Hosoda, J. 1986; 49 (1): 251-258

    Abstract

    Low dose cryoelectron microscopy has been used to record images and electron diffraction patterns of frozen hydrated crystals of the single-stranded DNA binding protein gp32*I. Fourier transforms from 13 image areas, corresponding to approximately 40,000 unit cells, were aligned by a minimal phase residual search and merged by vector addition in reciprocal space. Phases from the resulting composite transform were combined with amplitudes from electron diffraction patterns to reconstruct the projected mass density of the gp32*I crystal at 8.4 A resolution.

    View details for Web of Science ID A1986AYX5300072

    View details for PubMedID 3513856

  • STRUCTURAL-ANALYSIS OF T4 DNA HELIX DESTABILIZING PROTEIN (GP32-STAR-I) CRYSTAL BY ELECTRON-MICROSCOPY JOURNAL OF MOLECULAR BIOLOGY Cohen, H., Chiu, W., Cohen, H. A., Chiu, W. 1983; 169 (1): 235-248
  • STRUCTURE OF THE SURFACE-LAYER PROTEIN OF THE OUTER-MEMBRANE OF SPIRILLUM-SERPENS JOURNAL OF ULTRASTRUCTURE RESEARCH Glaeser, R. M., Chiu, W., Grano, D. 1979; 66 (3): 235-242

    View details for Web of Science ID A1979GU49700003

    View details for PubMedID 439191

  • FACTORS AFFECTING HIGH-RESOLUTION FIXED-BEAM TRANSMISSION ELECTRON-MICROSCOPY ULTRAMICROSCOPY Chiu, W., Glaeser, R. M. 1977; 2 (2-3): 207-217

    Abstract

    An experimental and theoretical characterization of a fixed-beam transmission electron microscope with a field emission gun has been made with regard to the factors of electron beam brightness, spatial and temporal coherence of the incident electrons, objective lens current fluctuation, mechanical stability, and specimen contamination. It has been found that mechanical stability and temporal coherence are the primary factors that prevent the contrast transfer function from extending to 2.0 A in our microscope. Different amorphous thin films have also been used in order to compare their suitability for testing the imaging capability of the microscope at atomic resolution.

    View details for Web of Science ID A1977DT14100011

    View details for PubMedID 888240

  • SINGLE ATOM IMAGE-CONTRAST - CONVENTIONAL DARK-FIELD AND BRIGHT-FIELD ELECTRON-MICROSCOPY JOURNAL OF MICROSCOPY-OXFORD Chiu, W., Glaeser, R. M. 1975; 103 (JAN): 33-54