York University, B.Sc. 1995-1999, Computer Science - Computer Graphics, HCI
University of Toronto, M.Sc. 1999-2001, Computer Science, Computer Graphics
Blueprint Initiative, 2001-2005 - Bioinformatics Research
MIT, Ph.D. 2005-2011 - Electrical Engineering and Computer Science, Biology - CryoEM map segmentation and fitting of atomic models
Baylor College of Medicine 2011-2017 - Scientific Programmer - Cryo-EM map analysis and atomic modeling
Stanford University 2017-present - Research Scientist - Cryo-EM map analysis and atomic modeling

Academic Appointments

  • Basic Life Science Research Associate, Bioengineering

All Publications

  • Three-dimensional structure-guided evolution of a ribosome with tethered subunits. Nature chemical biology Kim, D. S., Watkins, A., Bidstrup, E., Lee, J., Topkar, V., Kofman, C., Schwarz, K. J., Liu, Y., Pintilie, G., Roney, E., Das, R., Jewett, M. C. 2022


    RNA-based macromolecular machines, such as the ribosome, have functional parts reliant on structural interactions spanning sequence-distant regions. These features limit evolutionary exploration of mutant libraries and confound three-dimensional structure-guided design. To address these challenges, we describe Evolink (evolution and linkage), a method that enables high-throughput evolution of sequence-distant regions in large macromolecular machines, and library design guided by computational RNA modeling to enable exploration of structurally stable designs. Using Evolink, we evolved a tethered ribosome with a 58% increased activity in orthogonal protein translation and a 97% improvement in doubling times in SQ171 cells compared to a previously developed tethered ribosome, and reveal new permissible sequences in a pair of ribosomal helices with previously explored biological function. The Evolink approach may enable enhanced engineering of macromolecular machines for new and improved functions for synthetic biology.

    View details for DOI 10.1038/s41589-022-01064-w

    View details for PubMedID 35836020

  • Cryo-EM analysis of Ebola virus nucleocapsid-like assembly. STAR protocols Wang, Y., Binning, J. M., Pintilie, G. D., Chiu, W., Amarasinghe, G. K., Leung, D. W., Su, Z. 1800; 3 (1): 101030


    This protocol describes the reconstitution of the filamentous Ebola virus nucleocapsid-like assembly in vitro. This is followed by solving the cryo-EM structure using helical reconstruction, and flexible fitting of the existing model into the 5.8 A cryo-EM map. The protocol can be applied to other filamentous viral protein assemblies, particularly those with high flexibility and moderate resolution maps, which present technical challenges to model building. For complete details on the use and execution of this profile, please refer to Su etal. (2018).

    View details for DOI 10.1016/j.xpro.2021.101030

    View details for PubMedID 34977676

  • Cryo-EM, Protein Engineering, and Simulation Enable the Development of Peptide Therapeutics against Acute Myeloid Leukemia. ACS central science Zhang, K., Horikoshi, N., Li, S., Powers, A. S., Hameedi, M. A., Pintilie, G. D., Chae, H., Khan, Y. A., Suomivuori, C., Dror, R. O., Sakamoto, K. M., Chiu, W., Wakatsuki, S. 2022; 8 (2): 214-222


    Cryogenic electron microscopy (cryo-EM) has emerged as a viable structural tool for molecular therapeutics development against human diseases. However, it remains a challenge to determine structures of proteins that are flexible and smaller than 30 kDa. The 11 kDa KIX domain of CREB-binding protein (CBP), a potential therapeutic target for acute myeloid leukemia and other cancers, is a protein which has defied structure-based inhibitor design. Here, we develop an experimental approach to overcome the size limitation by engineering a protein double-shell to sandwich the KIX domain between apoferritin as the inner shell and maltose-binding protein as the outer shell. To assist homogeneous orientations of the target, disulfide bonds are introduced at the target-apoferritin interface, resulting in a cryo-EM structure at 2.6 A resolution. We used molecular dynamics simulations to design peptides that block the interaction of the KIX domain of CBP with the intrinsically disordered pKID domain of CREB. The double-shell design allows for fluorescence polarization assays confirming the binding between the KIX domain in the double-shell and these interacting peptides. Further cryo-EM analysis reveals a helix-helix interaction between a single KIX helix and the best peptide, providing a possible strategy for developments of next-generation inhibitors.

    View details for DOI 10.1021/acscentsci.1c01090

    View details for PubMedID 35233453

  • Cryo-ET of Toxoplasma parasites gives subnanometer insight into tubulin-based structures. Proceedings of the National Academy of Sciences of the United States of America Sun, S. Y., Segev-Zarko, L., Chen, M., Pintilie, G. D., Schmid, M. F., Ludtke, S. J., Boothroyd, J. C., Chiu, W. 2022; 119 (6)


    Tubulin is a conserved protein that polymerizes into different forms of filamentous structures in Toxoplasma gondii, an obligate intracellular parasite in the phylum Apicomplexa. Two key tubulin-containing cytoskeletal components are subpellicular microtubules (SPMTs) and conoid fibrils (CFs). The SPMTs help maintain shape and gliding motility, while the CFs are implicated in invasion. Here, we use cryogenic electron tomography to determine the molecular structures of the SPMTs and CFs in vitrified intact and detergent-extracted parasites. Subvolume densities from detergent-extracted parasites yielded averaged density maps at subnanometer resolutions, and these were related back to their architecture in situ. An intralumenal spiral lines the interior of the 13-protofilament SPMTs, revealing a preferred orientation of these microtubules relative to the parasite's long axis. Each CF is composed of nine tubulin protofilaments that display a comma-shaped cross-section, plus additional associated components. Conoid protrusion, a crucial step in invasion, is associated with an altered pitch of each CF. The use of basic building blocks of protofilaments and different accessory proteins in one organism illustrates the versatility of tubulin to form two distinct types of assemblies, SPMTs and CFs.

    View details for DOI 10.1073/pnas.2111661119

    View details for PubMedID 35121661

  • Complete three-dimensional structures of the Lon protease translocating a protein substrate. Science advances Li, S., Hsieh, K., Kuo, C., Lee, S., Pintilie, G. D., Zhang, K., Chang, C. 2021; 7 (42): eabj7835


    [Figure: see text].

    View details for DOI 10.1126/sciadv.abj7835

    View details for PubMedID 34652947

  • Molecular basis for ATPase-powered substrate translocation by the Lon AAA+ protease. The Journal of biological chemistry Li, S., Hsieh, K., Su, S., Pintilie, G. D., Zhang, K., Chang, C. 2021: 101239


    The Lon AAA+ (adenosine triphosphatases associated with diverse cellular activities) protease (LonA) converts ATP-fuelled conformational changes into sufficient mechanical force to drive translocation of a substrate into a hexameric proteolytic chamber. To understand the structural basis for the substrate translocation process, we determined the cryo-electron microscopy (cryo-EM) structure of Meiothermus taiwanensis LonA (MtaLonA) in a substrate-engaged state at 3.6 A resolution. Our data indicate that substrate interactions are mediated by the dual pore-loops of the ATPase domains, organized in spiral staircase arrangement from four consecutive protomers in different ATP-binding and hydrolysis states. However, a closed AAA+ ring is maintained by two disengaged ADP-bound protomers transiting between the lowest and highest position. This structure reveals a processive rotary translocation mechanism mediated by LonA-specific nucleotide-dependent allosteric coordination among the ATPase domains, which is induced by substrate binding.

    View details for DOI 10.1016/j.jbc.2021.101239

    View details for PubMedID 34563541

  • Validation, analysis and annotation of cryo-EM structures. Acta crystallographica. Section D, Structural biology Pintilie, G., Chiu, W. 2021; 77 (Pt 9): 1142-1152


    The process of turning 2D micrographs into 3D atomic models of the imaged macromolecules has been under rapid development and scrutiny in the field of cryo-EM. Here, some important methods for validation at several stages in this process are described. Firstly, how Fourier shell correlation of two independent maps and phase randomization beyond a certain frequency address the assessment of map resolution is reviewed. Techniques for local resolution estimation and map sharpening are also touched upon. The topic of validating models which are either built de novo or based on a known atomic structure fitted into a cryo-EM map is then approached. Map-model comparison using Q-scores and Fourier shell correlation plots is used to assure the agreement of the model with the observed map density. The importance of annotating the model with B factors to account for the resolvability of individual atoms in the map is illustrated. Finally, the timely topic of detecting and validating water molecules and metal ions in maps that have surpassed 2 A resolution is described.

    View details for DOI 10.1107/S2059798321006069

    View details for PubMedID 34473085

  • Cryo-EM and antisense targeting of the 28-kDa frameshift stimulation element from the SARS-CoV-2 RNA genome. Nature structural & molecular biology Zhang, K., Zheludev, I. N., Hagey, R. J., Haslecker, R., Hou, Y. J., Kretsch, R., Pintilie, G. D., Rangan, R., Kladwang, W., Li, S., Wu, M. T., Pham, E. A., Bernardin-Souibgui, C., Baric, R. S., Sheahan, T. P., D'Souza, V., Glenn, J. S., Chiu, W., Das, R. 2021


    Drug discovery campaigns against COVID-19 are beginning to target the SARS-CoV-2 RNA genome. The highly conserved frameshift stimulation element (FSE), required for balanced expression of viral proteins, is a particularly attractive SARS-CoV-2 RNA target. Here we present a 6.9A resolution cryo-EM structure of the FSE (88nucleotides, ~28kDa), validated through an RNA nanostructure tagging method. The tertiary structure presents a topologically complex fold in which the 5' end is threaded through a ring formed inside a three-stem pseudoknot. Guided by this structure, we develop antisense oligonucleotides that impair FSE function in frameshifting assays and knock down SARS-CoV-2 virus replication in A549-ACE2 cells at 100nM concentration.

    View details for DOI 10.1038/s41594-021-00653-y

    View details for PubMedID 34426697

  • Quantifiying resolvability of atomic features in cryo-EM maps using Q-scores Pintilie, G., Schmid, M. F., Chiu, W. INT UNION CRYSTALLOGRAPHY. 2021: C58
  • Evolution of Standardization and Dissemination of Cryo-EM Structures and Data Jointly by the Community, PDB and EMDB. The Journal of biological chemistry Chiu, W., Schmid, M. F., Pintilie, G., Lawson, C. L. 2021: 100560


    Cryogenic electron microscopy (cryo-EM) methods began to be used in the mid-1970s to study thin and periodic arrays of proteins. Following a half-century of development in cryo-specimen preparation, instrumentation, data collection, data processing and modeling software, cryo-EM has become a routine method for solving structures from large biological assemblies to small biomolecules at near to true atomic resolution. This review explores the critical roles played by the Protein Data Bank (PDB) and Electron Microscopy Data Bank (EMDB) in partnership with the community to develop the necessary infrastructure to archive cryo-EM maps and associated models. Public access to cryo-EM structure data has in turn facilitated better understanding of structure-function relationships and advancement of image processing and modeling tool development. The partnership between the global cryo-EM community and PDB and EMDB leadership has synergistically shaped the standards for metadata, one-stop deposition of maps and models, and validation metrics to assess the quality of cryo-EM structures. The advent of cryo-electron tomography (cryo-ET) for in situ molecular cell structures at a broad resolution range and their correlations with other imaging data introduces new data archival challenges in terms of data size and complexity in the years to come.

    View details for DOI 10.1016/j.jbc.2021.100560

    View details for PubMedID 33744287

  • Cryo-EM model validation recommendations based on outcomes of the 2019 EMDataResource challenge. Nature methods Lawson, C. L., Kryshtafovych, A., Adams, P. D., Afonine, P. V., Baker, M. L., Barad, B. A., Bond, P., Burnley, T., Cao, R., Cheng, J., Chojnowski, G., Cowtan, K., Dill, K. A., DiMaio, F., Farrell, D. P., Fraser, J. S., Herzik, M. A., Hoh, S. W., Hou, J., Hung, L., Igaev, M., Joseph, A. P., Kihara, D., Kumar, D., Mittal, S., Monastyrskyy, B., Olek, M., Palmer, C. M., Patwardhan, A., Perez, A., Pfab, J., Pintilie, G. D., Richardson, J. S., Rosenthal, P. B., Sarkar, D., Schafer, L. U., Schmid, M. F., Schroder, G. F., Shekhar, M., Si, D., Singharoy, A., Terashi, G., Terwilliger, T. C., Vaiana, A., Wang, L., Wang, Z., Wankowicz, S. A., Williams, C. J., Winn, M., Wu, T., Yu, X., Zhang, K., Berman, H. M., Chiu, W. 2021; 18 (2): 156–64


    This paper describes outcomes of the 2019 Cryo-EM Model Challenge. The goals were to (1) assess the quality of models that can be produced from cryogenic electron microscopy (cryo-EM) maps using current modeling software, (2) evaluate reproducibility of modeling results from different software developers and users and (3) compare performance of current metrics used for model evaluation, particularly Fit-to-Map metrics, with focus on near-atomic resolution. Our findings demonstrate the relatively high accuracy and reproducibility of cryo-EM models derived by 13 participating teams from four benchmark maps, including three forming a resolution series (1.8 to 3.1A). The results permit specific recommendations to be made about validating near-atomic cryo-EM structures both in the context of individual experiments and structure data archives such as the Protein Data Bank. We recommend the adoption of multiple scoring parameters to provide full and objective annotation and assessment of the model, reflective of the observed cryo-EM map density.

    View details for DOI 10.1038/s41592-020-01051-w

    View details for PubMedID 33542514

  • The N-terminus of varicella-zoster virus glycoprotein B has a functional role in fusion. PLoS pathogens Oliver, S. L., Xing, Y. n., Chen, D. H., Roh, S. H., Pintilie, G. D., Bushnell, D. A., Sommer, M. H., Yang, E. n., Carfi, A. n., Chiu, W. n., Arvin, A. M. 2021; 17 (1): e1008961


    Varicella-zoster virus (VZV) is a medically important alphaherpesvirus that induces fusion of the virion envelope and the cell membrane during entry, and between cells to form polykaryocytes within infected tissues during pathogenesis. All members of the Herpesviridae, including VZV, have a conserved core fusion complex composed of glycoproteins, gB, gH and gL. The ectodomain of the primary fusogen, gB, has five domains, DI-V, of which DI contains the fusion loops needed for fusion function. We recently demonstrated that DIV is critical for fusion initiation, which was revealed by a 2.8Å structure of a VZV neutralizing mAb, 93k, bound to gB and mutagenesis of the gB-93k interface. To further assess the mechanism of mAb 93k neutralization, the binding site of a non-neutralizing mAb to gB, SG2, was compared to mAb 93k using single particle cryogenic electron microscopy (cryo-EM). The gB-SG2 interface partially overlapped with that of gB-93k but, unlike mAb 93k, mAb SG2 did not interact with the gB N-terminus, suggesting a potential role for the gB N-terminus in membrane fusion. The gB ectodomain structure in the absence of antibody was defined at near atomic resolution by single particle cryo-EM (3.9Å) of native full-length gB purified from infected cells and by X-ray crystallography (2.4Å) of the transiently expressed ectodomain. Both structures revealed that the VZV gB N-terminus (aa72-114) was flexible based on the absence of visible structures in the cryo-EM or X-ray crystallography data but the presence of gB N-terminal peptides were confirmed by mass spectrometry. Notably, N-terminal residues 109KSQD112 were predicted to form a small α-helix and alanine substitution of these residues abolished cell-cell fusion in a virus-free assay. Importantly, transferring the 109AAAA112 mutation into the VZV genome significantly impaired viral propagation. These data establish a functional role for the gB N-terminus in membrane fusion broadly relevant to the Herpesviridae.

    View details for DOI 10.1371/journal.ppat.1008961

    View details for PubMedID 33411789

  • Cryo-EM structures of full-length Tetrahymena ribozyme at 3.1 Å resolution. Nature Su, Z., Zhang, K., Kappel, K., Li, S., Palo, M. Z., Pintilie, G. D., Rangan, R., Luo, B., Wei, Y., Das, R., Chiu, W. 2021


    Single-particle cryogenic electron microscopy (cryo-EM) has become a standard technique for determining protein structures at atomic resolution1-3. However, cryo-EM studies of protein-free RNA are in their early days. The Tetrahymena thermophila group I self-splicing intron was the first ribozyme to be discovered and has been a prominent model system for the study of RNA catalysis and structure-function relationships4, but its full structure remains unknown. Here we report cryo-EM structures of the full-length Tetrahymena ribozyme in substrate-free and bound states at a resolution of 3.1 Å. Newly resolved peripheral regions form two coaxially stacked helices; these are interconnected by two kissing loop pseudoknots that wrap around the catalytic core and include two previously unforeseen (to our knowledge) tertiary interactions. The global architecture is nearly identical in both states; only the internal guide sequence and guanosine binding site undergo a large conformational change and a localized shift, respectively, upon binding of RNA substrates. These results provide a long-sought structural view of a paradigmatic RNA enzyme and signal a new era for the cryo-EM-based study of structure-function relationships in ribozymes.

    View details for DOI 10.1038/s41586-021-03803-w

    View details for PubMedID 34381213

  • Mapping the catalytic conformations of an assembly-line polyketide synthase module. Science (New York, N.Y.) Cogan, D. P., Zhang, K., Li, X., Li, S., Pintilie, G. D., Roh, S. H., Craik, C. S., Chiu, W., Khosla, C. 2021; 374 (6568): 729-734


    [Figure: see text].

    View details for DOI 10.1126/science.abi8358

    View details for PubMedID 34735239

  • Resolving individualatoms of protein complex by cryo-electron microscopy. Cell research Zhang, K., Pintilie, G. D., Li, S., Schmid, M. F., Chiu, W. 2020

    View details for DOI 10.1038/s41422-020-00432-2

    View details for PubMedID 33139928

  • Publisher Correction: A glycoprotein B-neutralizing antibody structure at 2.8 A uncovers a critical domain for herpesvirus fusion initiation. Nature communications Oliver, S. L., Xing, Y., Chen, D., Roh, S. H., Pintilie, G. D., Bushnell, D. A., Sommer, M. H., Yang, E., Carfi, A., Chiu, W., Arvin, A. M. 2020; 11 (1): 4398


    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

    View details for DOI 10.1038/s41467-020-18385-w

    View details for PubMedID 32859924

  • A glycoprotein B-neutralizing antibody structure at 2.8A uncovers a critical domain for herpesvirus fusion initiation. Nature communications Oliver, S. L., Xing, Y., Chen, D., Roh, S. H., Pintilie, G. D., Bushnell, D. A., Sommer, M. H., Yang, E., Carfi, A., Chiu, W., Arvin, A. M. 2020; 11 (1): 4141


    Members of the Herpesviridae, including the medically important alphaherpesvirus varicella-zoster virus (VZV), induce fusion of the virion envelope with cell membranes during entry, and between cells to form polykaryocytes in infected tissues. The conserved glycoproteins, gB, gH and gL, are the core functional proteins of the herpesvirus fusion complex. gB serves as the primary fusogen via its fusion loops, but functions for the remaining gB domains remain unexplained. As a pathway for biological discovery of domain function, our approach used structure-based analysis of the viral fusogen together with a neutralizing antibody. We report here a 2.8A cryogenic-electron microscopy structure of native gB recovered from VZV-infected cells, in complex with a human monoclonal antibody, 93k. This high-resolution structure guided targeted mutagenesis at the gB-93k interface, providing compelling evidence that a domain spatially distant from the gB fusion loops is critical for herpesvirus fusion, revealing a potential new target for antiviral therapies.

    View details for DOI 10.1038/s41467-020-17911-0

    View details for PubMedID 32811830

  • 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


    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

  • 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


    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

  • Full-length three-dimensional structure of the influenza A virus M1 protein and its organization into a matrix layer. PLoS biology Selzer, L. n., Su, Z. n., Pintilie, G. D., Chiu, W. n., Kirkegaard, K. n. 2020; 18 (9): e3000827


    Matrix proteins are encoded by many enveloped viruses, including influenza viruses, herpes viruses, and coronaviruses. Underneath the viral envelope of influenza virus, matrix protein 1 (M1) forms an oligomeric layer critical for particle stability and pH-dependent RNA genome release. However, high-resolution structures of full-length monomeric M1 and the matrix layer have not been available, impeding antiviral targeting and understanding of the pH-dependent transitions involved in cell entry. Here, purification and extensive mutagenesis revealed protein-protein interfaces required for the formation of multilayered helical M1 oligomers similar to those observed in virions exposed to the low pH of cell entry. However, single-layered helical oligomers with biochemical and ultrastructural similarity to those found in infectious virions before cell entry were observed upon mutation of a single amino acid. The highly ordered structure of the single-layered oligomers and their likeness to the matrix layer of intact virions prompted structural analysis by cryo-electron microscopy (cryo-EM). The resulting 3.4-Å-resolution structure revealed the molecular details of M1 folding and its organization within the single-shelled matrix. The solution of the full-length M1 structure, the identification of critical assembly interfaces, and the development of M1 assembly assays with purified proteins are crucial advances for antiviral targeting of influenza viruses.

    View details for DOI 10.1371/journal.pbio.3000827

    View details for PubMedID 32997652

  • A 3.4-Å cryo-electron microscopy structure of the human coronavirus spike trimer computationally derived from vitrified NL63 virus particles. QRB discovery Zhang, K., Li, S., Pintilie, G., Chmielewski, D., Schmid, M. F., Simmons, G., Jin, J., Chiu, W. 2020; 1: e11


    Human coronavirus NL63 (HCoV-NL63) is an enveloped pathogen of the family Coronaviridae that spreads worldwide and causes up to 10% of all annual respiratory diseases. HCoV-NL63 is typically associated with mild upper respiratory symptoms in children, elderly and immunocompromised individuals. It has also been shown to cause severe lower respiratory illness. NL63 shares ACE2 as a receptor for viral entry with SARS-CoV-1 and SARS-CoV-2. Here, we present the in situ structure of HCoV-NL63 spike (S) trimer at 3.4-Å resolution by single-particle cryo-EM imaging of vitrified virions without chemical fixative. It is structurally homologous to that obtained previously from the biochemically purified ectodomain of HCoV-NL63 S trimer, which displays a three-fold symmetric trimer in a single conformation. In addition to previously proposed and observed glycosylation sites, our map shows density at other sites, as well as different glycan structures. The domain arrangement within a protomer is strikingly different from that of the SARS-CoV-2 S and may explain their different requirements for activating binding to the receptor. This structure provides the basis for future studies of spike proteins with receptors, antibodies or drugs, in the native state of the coronavirus particles.

    View details for DOI 10.1017/qrd.2020.16

    View details for PubMedID 34192263

    View details for PubMedCentralID PMC7737156

  • Cryo-EM and MD infer water-mediated proton transport and autoinhibition mechanisms of Vo complex. Science advances Roh, S. H., Shekhar, M. n., Pintilie, G. n., Chipot, C. n., Wilkens, S. n., Singharoy, A. n., Chiu, W. n. 2020; 6 (41)


    Rotary vacuolar adenosine triphosphatases (V-ATPases) drive transmembrane proton transport through a Vo proton channel subcomplex. Despite recent high-resolution structures of several rotary ATPases, the dynamic mechanism of proton pumping remains elusive. Here, we determined a 2.7-Å cryo-electron microscopy (cryo-EM) structure of yeast Vo proton channel in nanodisc that reveals the location of ordered water molecules along the proton path, details of specific protein-lipid interactions, and the architecture of the membrane scaffold protein. Moreover, we uncover a state of Vo that shows the c-ring rotated by ~14°. Molecular dynamics simulations demonstrate that the two rotary states are in thermal equilibrium and depict how the protonation state of essential glutamic acid residues couples water-mediated proton transfer with c-ring rotation. Our cryo-EM models and simulations also rationalize a mechanism for inhibition of passive proton transport as observed for free Vo that is generated as a result of V-ATPase regulation by reversible disassembly in vivo.

    View details for DOI 10.1126/sciadv.abb9605

    View details for PubMedID 33028525

  • Accelerated cryo-EM-guided determination of three-dimensional RNA-only structures. Nature methods Kappel, K. n., Zhang, K. n., Su, Z. n., Watkins, A. M., Kladwang, W. n., Li, S. n., Pintilie, G. n., Topkar, V. V., Rangan, R. n., Zheludev, I. N., Yesselman, J. D., Chiu, W. n., Das, R. n. 2020; 17 (7): 699–707


    The discovery and design of biologically important RNA molecules is outpacing three-dimensional structural characterization. Here, we demonstrate that cryo-electron microscopy can routinely resolve maps of RNA-only systems and that these maps enable subnanometer-resolution coordinate estimation when complemented with multidimensional chemical mapping and Rosetta DRRAFTER computational modeling. This hybrid 'Ribosolve' pipeline detects and falsifies homologies and conformational rearrangements in 11 previously unknown 119- to 338-nucleotide protein-free RNA structures: full-length Tetrahymena ribozyme, hc16 ligase with and without substrate, full-length Vibrio cholerae and Fusobacterium nucleatum glycine riboswitch aptamers with and without glycine, Mycobacterium SAM-IV riboswitch with and without S-adenosylmethionine, and the computer-designed ATP-TTR-3 aptamer with and without AMP. Simulation benchmarks, blind challenges, compensatory mutagenesis, cross-RNA homologies and internal controls demonstrate that Ribosolve can accurately resolve the global architectures of RNA molecules but does not resolve atomic details. These tests offer guidelines for making inferences in future RNA structural studies with similarly accelerated throughput.

    View details for DOI 10.1038/s41592-020-0878-9

    View details for PubMedID 32616928

  • 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


    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

  • 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-+


    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

  • Rapid RNA structure determination through cryo-EM, high-throughput biochemistry, and computational modeling Kappel, K., Zhang, K., Su, Z., Pintilie, G., Chiu, W., Das, R. AMER CHEMICAL SOC. 2019
  • The Chaperonin TRiC/CCT Associates with Prefoldin through a Conserved Electrostatic Interface Essential for Cellular Proteostasis CELL Gestaut, D., Roh, S., Ma, B., Pintilie, G., Joachimiak, L. A., Leitner, A., Walzthoeni, T., Aebersold, R., Chiu, W., Frydman, J. 2019; 177 (3): 751-+
  • 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 Bedeme, H., Schmid, M. F., Au, S., Chiu, W. 2019; 116 (14): 6800–6805
  • 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


    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


    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


    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
  • Evolving Data Standards for cryo Electron Microscopy Lawson, C. L., Kryshtafovych, A., Pintilie, G., Berman, H. M., Chiu, W. INT UNION CRYSTALLOGRAPHY. 2019: A68
  • Cryo-EM structure of a 40 kDa SAM-IV riboswitch RNA at 3.7 Å resolution. Nature communications Zhang, K. n., Li, S. n., Kappel, K. n., Pintilie, G. n., Su, Z. n., Mou, T. C., Schmid, M. F., Das, R. n., Chiu, W. n. 2019; 10 (1): 5511


    Specimens below 50 kDa have generally been considered too small to be analyzed by single-particle cryo-electron microscopy (cryo-EM). The high flexibility of pure RNAs makes it difficult to obtain high-resolution structures by cryo-EM. In bacteria, riboswitches regulate sulfur metabolism through binding to the S-adenosylmethionine (SAM) ligand and offer compelling targets for new antibiotics. SAM-I, SAM-I/IV, and SAM-IV are the three most commonly found SAM riboswitches, but the structure of SAM-IV is still unknown. Here, we report the structures of apo and SAM-bound SAM-IV riboswitches (119-nt, ~40 kDa) to 3.7 Å and 4.1 Å resolution, respectively, using cryo-EM. The structures illustrate homologies in the ligand-binding core but distinct peripheral tertiary contacts in SAM-IV compared to SAM-I and SAM-I/IV. Our results demonstrate the feasibility of resolving small RNAs with enough detail to enable detection of their ligand-binding pockets and suggest that cryo-EM could play a role in structure-assisted drug design for RNA.

    View details for DOI 10.1038/s41467-019-13494-7

    View details for PubMedID 31796736

  • 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


    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

  • 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


    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

  • Comparison of Crystal and Cryoem Structures of Hsp104 and ClpB Disaggregases Joachimiak, A., Michalska, K., Zhang, K., March, Z., Hatzos-Skintges, C., Pintilie, G., Bigelow, L., Castellano, L., Miles, L., Jackrel, M., Chuang, E., Jedrzejczak, R., Shorter, J., Chiu, W. WILEY. 2018: 32–33
  • 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


    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

  • The 3.5-A CryoEM Structure of Nanodisc-Reconstituted Yeast Vacuolar ATPase Vo Proton Channel The 3.5-A CryoEM Structure of Nanodisc-Reconstituted Yeast Vacuolar ATPase Vo Proton Channel Roh, S., Stam, N. J., Hryc, C. F., Couoh-Cardel,, S., Pintilie, G., Chiu, W., Wilkens, S. 2018; 69 (6): 993-1004


    The molecular mechanism of transmembrane proton translocation in rotary motor ATPases is not fully understood. Here, we report the 3.5-Å resolution cryoEM structure of the lipid nanodisc-reconstituted Vo proton channel of the yeast vacuolar H+-ATPase, captured in a physiologically relevant, autoinhibited state. The resulting atomic model provides structural detail for the amino acids that constitute the proton pathway at the interface of the proteolipid ring and subunit a. Based on the structure and previous mutagenesis studies, we propose the chemical basis of transmembrane proton transport. Moreover, we discovered that the C terminus of the assembly factor Voa1 is an integral component of mature Vo. Voa1's C-terminal transmembrane α helix is bound inside the proteolipid ring, where it contributes to the stability of the complex. Our structure rationalizes possible mechanisms by which mutations in human Vo can result in disease phenotypes and may thus provide new avenues for therapeutic interventions.

    View details for DOI 10.1016/j.molcel.2018.02.006

    View details for PubMedCentralID PMC5893162

  • 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


    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

  • 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


    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 Web of Science ID 000370763800011

    View details for PubMedID 26743049

    View details for PubMedCentralID PMC4775875