Muyuan Chen

All Publications

• Centriole and transition zone structures in photoreceptor cilia revealed by cryo-electron tomography. Life science alliance Zhang, Z., Moye, A. R., He, F., Chen, M., Agosto, M. A., Wensel, T. G. 2024; 7 (3)

  Abstract

  Primary cilia mediate sensory signaling in multiple organisms and cell types but have structures adapted for specific roles. Structural defects in them lead to devastating diseases known as ciliopathies in humans. Key to their functions are structures at their base: the basal body, the transition zone, the "Y-shaped links," and the "ciliary necklace." We have used cryo-electron tomography with subtomogram averaging and conventional transmission electron microscopy to elucidate the structures associated with the basal region of the "connecting cilia" of rod outer segments in mouse retina. The longitudinal variations in microtubule (MT) structures and the lumenal scaffold complexes connecting them have been determined, as well as membrane-associated transition zone structures: Y-shaped links connecting MT to the membrane, and ciliary beads connected to them that protrude from the cell surface and form a necklace-like structure. These results represent a clearer structural scaffold onto which molecules identified by genetics, proteomics, and superresolution fluorescence can be placed in our emerging model of photoreceptor sensory cilia.

  View details for DOI 10.26508/lsa.202302409

  View details for PubMedID 38182160

• Genome organization in double-stranded DNA viruses observed by cryoET. bioRxiv : the preprint server for biology Chen, M., Sahoo, B., Mou, Z., Song, X., Tsai, T., Dai, X. 2023

  Abstract

  Double-stranded DNA (dsDNA) viruses package their genetic material into protein cages with diameters usually a few hundred times smaller than the length of their genome. Compressing the relatively stiff and highly negatively charged dsDNA into a small volume is energetically costly and mechanistically enigmatic. Multiple models of dsDNA packaging have been proposed based on various experimental evidence and simulation methods, but direct observation of any viral genome organization is lacking. Here, using cryoET and an improved data processing scheme that utilizes information from the encaging protein shell, we present 3D views of dsDNA genome inside individual viral particles at resolution that densities of neighboring DNA duplexes are readily separable. These cryoET observations reveal a "rod-and-coil" fold of the dsDNA that is conserved among herpes simplex virus type 1 (HSV-1) with a spherical capsid, bacteriophage T4 with a prolate capsid, and bacteriophage T7 with a proteinaceous core inside the capsid. Finally, inspired by the genome arrangement in partially packaged T4 particles, we propose a mechanism for the genome packaging process in dsDNA viruses.

  View details for DOI 10.1101/2023.12.15.571939

  View details for PubMedID 38168199

  View details for PubMedCentralID PMC10760162

• Targeted mutagenesis of the herpesvirus fusogen central helix captures transition states. Nature communications Zhou, M., Vollmer, B., Machala, E., Chen, M., Grünewald, K., Arvin, A. M., Chiu, W., Oliver, S. L. 2023; 14 (1): 7958

  Abstract

  Herpesviruses remain a burden for animal and human health, including the medically important varicella-zoster virus (VZV). Membrane fusion mediated by conserved core glycoproteins, the fusogen gB and the heterodimer gH-gL, enables herpesvirus cell entry. The ectodomain of gB orthologs has five domains and is proposed to transition from a prefusion to postfusion conformation but the functional relevance of the domains for this transition remains poorly defined. Here we describe structure-function studies of the VZV gB DIII central helix targeting residues 526EHV528. Critically, a H527P mutation captures gB in a prefusion conformation as determined by cryo-EM, a loss of membrane fusion in a virus free assay, and failure of recombinant VZV to spread in cell monolayers. Importantly, two predominant cryo-EM structures of gB[H527P] are identified by 3D classification and focused refinement, suggesting they represented gB conformations in transition. These studies reveal gB DIII as a critical element for herpesvirus gB fusion function.

  View details for DOI 10.1038/s41467-023-43011-w

  View details for PubMedID 38042814

  View details for PubMedCentralID PMC10693595

• Improving resolution and resolvability of single-particle cryoEM structures using Gaussian mixture models. Nature methods Chen, M., Schmid, M. F., Chiu, W. 2023

  Abstract

  Cryogenic electron microscopy is widely used in structural biology, but its resolution is often limited by the dynamics of the macromolecule. Here we developed a refinement protocol based on Gaussian mixture models that integrates particle orientation and conformation estimation and improves the alignment for flexible domains of protein structures. We demonstrated this protocol on multiple datasets, resulting in improved resolution and resolvability, locally and globally, by visual and quantitative measures.

  View details for DOI 10.1038/s41592-023-02082-9

  View details for PubMedID 37973972

  View details for PubMedCentralID 6460916

• Structural insights into the modulation of coronavirus spike tilting and infectivity by hinge glycans. Nature communications Chmielewski, D., Wilson, E. A., Pintilie, G., Zhao, P., Chen, M., Schmid, M. F., Simmons, G., Wells, L., Jin, J., Singharoy, A., Chiu, W. 2023; 14 (1): 7175

  Abstract

  Coronavirus spike glycoproteins presented on the virion surface mediate receptor binding, and membrane fusion during virus entry and constitute the primary target for vaccine and drug development. How the structure dynamics of the full-length spikes incorporated in viral lipid envelope correlates with the virus infectivity remains poorly understood. Here we present structures and distributions of native spike conformations on vitrified human coronavirus NL63 (HCoV-NL63) virions without chemical fixation by cryogenic electron tomography (cryoET) and subtomogram averaging, along with site-specific glycan composition and occupancy determined by mass spectrometry. The higher oligomannose glycan shield on HCoV-NL63 spikes than on SARS-CoV-2 spikes correlates with stronger immune evasion of HCoV-NL63. Incorporation of cryoET-derived native spike conformations into all-atom molecular dynamic simulations elucidate the conformational landscape of the glycosylated, full-length spike that reveals a role of hinge glycans in modulating spike bending. We show that glycosylation at N1242 at the upper portion of the stalk is responsible for the extensive orientational freedom of the spike crown. Subsequent infectivity assays implicated involvement of N1242-glyan in virus entry. Our results suggest a potential therapeutic target site for HCoV-NL63.

  View details for DOI 10.1038/s41467-023-42836-9

  View details for PubMedID 37935678

  View details for PubMedCentralID PMC10630519

• Author Correction: Membrane translocation process revealed by in situ structures of type II secretion system secretins. Nature communications Yu, Z., Wu, Y., Chen, M., Huo, T., Zheng, W., Ludtke, S. J., Shi, X., Wang, Z. 2023; 14 (1): 4833

  View details for DOI 10.1038/s41467-023-40656-5

  View details for PubMedID 37563200

• Membrane translocation process revealed by in situ structures of type II secretion system secretins. Nature communications Yu, Z., Wu, Y., Chen, M., Huo, T., Zheng, W., Ludtke, S. J., Shi, X., Wang, Z. 2023; 14 (1): 4025

  Abstract

  The GspD secretin is the outer membrane channel of the bacterial type II secretion system (T2SS) which secrets diverse toxins that cause severe diseases such as diarrhea and cholera. GspD needs to translocate from the inner to the outer membrane to exert its function, and this process is an essential step for T2SS to assemble. Here, we investigate two types of secretins discovered so far in Escherichia coli, GspDalpha, and GspDbeta. By electron cryotomography subtomogram averaging, we determine in situ structures of key intermediate states of GspDalpha and GspDbeta in the translocation process, with resolution ranging from 9A to 19A. In our results, GspDalpha and GspDbeta present entirely different membrane interaction patterns and ways of transitioning the peptidoglycan layer. From this, we hypothesize two distinct models for the membrane translocation of GspDalpha and GspDbeta, providing a comprehensive perspective on the inner to outer membrane biogenesis of T2SS secretins.

  View details for DOI 10.1038/s41467-023-39583-2

  View details for PubMedID 37419909

• Integrating Molecular Models Into CryoEM Heterogeneity Analysis Using Scalable High-resolution Deep Gaussian Mixture Models JOURNAL OF MOLECULAR BIOLOGY Chen, M., Toader, B., Lederman, R. 2023; 435 (9): 168014

  Abstract

  Resolving the structural variability of proteins is often key to understanding the structure-function relationship of those macromolecular machines. Single particle analysis using Cryogenic electron microscopy (CryoEM), combined with machine learning algorithms, provides a way to reveal the dynamics within the protein system from noisy micrographs. Here, we introduce an improved computational method that uses Gaussian mixture models for protein structure representation and deep neural networks for conformation space embedding. By integrating information from molecular models into the heterogeneity analysis, we can analyze continuous protein conformational changes using structural information at the frequency of 1/3 Å-1, and present the results in a more interpretable form.

  View details for DOI 10.1016/j.jmb.2023.168014

  View details for Web of Science ID 000999046500001

  View details for PubMedID 36806476

  View details for PubMedCentralID PMC10164680

• Integrated analyses reveal a hinge glycan regulates coronavirus spike tilting and virus infectivity. Research square Chmielewski, D., Wilson, E. A., Pintilie, G., Zhao, P., Chen, M., Schmid, M. F., Simmons, G., Wells, L., Jin, J., Singharoy, A., Chiu, W. 2023

  Abstract

  Coronavirus spike glycoproteins presented on the virion surface mediate receptor binding, and membrane fusion during virus entry and constitute the primary target for vaccine and drug development. How the structure dynamics of the full-length spikes incorporated in viral lipid envelope correlates with the virus infectivity remains poorly understood. Here we present structures and distributions of native spike conformations on vitrified human coronavirus NL63 (HCoV-NL63) virions without chemical fixation by cryogenic electron tomography (cryoET) and subtomogram averaging, along with site-specific glycan composition and occupancy determined by mass spectroscopy. The higher oligomannose glycan shield on HCoV-NL63 spikes than on SARS-CoV-2 spikes correlates with stronger immune evasion of HCoV-NL63. Incorporation of cryoET-derived native spike conformations into all-atom molecular dynamic simulations elucidate the conformational landscape of the glycosylated, full-length spike that reveals a novel role of stalk glycans in modulating spike bending. We show that glycosylation at N1242 at the upper portion of the stalk is responsible for the extensive orientational freedom of the spike crown. Subsequent infectivity assays support the hypothesis that this glycan-dependent motion impacts virus entry. Our results suggest a potential therapeutic target site for HCoV-NL63.

  View details for DOI 10.21203/rs.3.rs-2553619/v1

  View details for PubMedID 36824920

  View details for PubMedCentralID PMC9949256

• Molecular architecture of the Chikungunya virus replication complex. Science advances Tan, Y. B., Chmielewski, D., Law, M. C., Zhang, K., He, Y., Chen, M., Jin, J., Luo, D. 2022; 8 (48): eadd2536

  Abstract

  To better understand how positive-strand (+) RNA viruses assemble membrane-associated replication complexes (RCs) to synthesize, process, and transport viral RNA in virus-infected cells, we determined both the high-resolution structure of the core RNA replicase of chikungunya virus and the native RC architecture in its cellular context at subnanometer resolution, using in vitro reconstitution and in situ electron cryotomography, respectively. Within the core RNA replicase, the viral polymerase nsP4, which is in complex with nsP2 helicase-protease, sits in the central pore of the membrane-anchored nsP1 RNA-capping ring. The addition of a large cytoplasmic ring next to the C terminus of nsP1 forms the holo-RNA-RC as observed at the neck of spherules formed in virus-infected cells. These results represent a major conceptual advance in elucidating the molecular mechanisms of RNA virus replication and the principles underlying the molecular architecture of RCs, likely to be shared with many pathogenic (+) RNA viruses.

  View details for DOI 10.1126/sciadv.add2536

  View details for PubMedID 36449616

• Conformational motions and ligand-binding underlying gating and regulation in IP3R channel. Nature communications Fan, G., Baker, M. R., Terry, L. E., Arige, V., Chen, M., Seryshev, A. B., Baker, M. L., Ludtke, S. J., Yule, D. I., Serysheva, I. I. 2022; 13 (1): 6942

  Abstract

  Inositol-1,4,5-trisphosphate receptors (IP3Rs) are activated by IP3 and Ca2+ and their gating is regulated by various intracellular messengers that finely tune the channel activity. Here, using single particle cryo-EM analysis we determined 3D structures of the nanodisc-reconstituted IP3R1 channel in two ligand-bound states. These structures provide unprecedented details governing binding of IP3, Ca2+ and ATP, revealing conformational changes that couple ligand-binding to channel opening. Using a deep-learning approach and 3D variability analysis we extracted molecular motions of the key protein domains from cryo-EM density data. We find that IP3 binding relies upon intrinsic flexibility of the ARM2 domain in the tetrameric channel. Our results highlight a key role of dynamic side chains in regulating gating behavior of IP3R channels. This work represents a stepping-stone to developing mechanistic understanding of conformational pathways underlying ligand-binding, activation and regulation of the channel.

  View details for DOI 10.1038/s41467-022-34574-1

  View details for PubMedID 36376291

  View details for PubMedCentralID PMC9663519

• 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)

  Abstract

  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

• In situ structure of the AcrAB-TolC efflux pump at subnanometer resolution. Structure (London, England : 1993) Chen, M., Shi, X., Yu, Z., Fan, G., Serysheva, I. I., Baker, M. L., Luisi, B. F., Ludtke, S. J., Wang, Z. 2022; 30 (1): 107-113.e3

  Abstract

  The tripartite AcrAB-TolC assembly, which spans both the inner and outer membranes in Gram-negative bacteria, is an efflux pump that contributes to multidrug resistance. Here, we present the in situ structure of full-length Escherichia coli AcrAB-TolC determined at 7 Å resolution by electron cryo-tomography. The TolC channel penetrates the outer membrane bilayer through to the outer leaflet and exhibits two different configurations that differ by a 60° rotation relative to the AcrB position in the pump assembly. AcrA protomers interact directly with the inner membrane and with AcrB via an interface located in proximity to the AcrB ligand-binding pocket. Our structural analysis suggests that these AcrA-bridged interactions underlie an allosteric mechanism for transmitting drug-evoked signals from AcrB to the TolC channel within the pump. Our study demonstrates the power of in situ electron cryo-tomography, which permits critical insights into the function of bacterial efflux pumps.

  View details for DOI 10.1016/j.str.2021.08.008

  View details for PubMedID 34506732

  View details for PubMedCentralID PMC8741639

• The structure-function relationship of a signaling-competent, dimeric Reelin fragment. Structure (London, England : 1993) Turk, L. S., Kuang, X., Dal Pozzo, V., Patel, K., Chen, M., Huynh, K., Currie, M. J., Mitchell, D., Dobson, R. C., D'Arcangelo, G., Dai, W., Comoletti, D. 2021; 29 (10): 1156-1170.e6

  Abstract

  Reelin operates through canonical and non-canonical pathways that mediate several aspects of brain development and function. Reelin's dimeric central fragment (CF), generated through proteolytic cleavage, is required for the lipoprotein-receptor-dependent canonical pathway activation. Here, we analyze the signaling properties of a variety of Reelin fragments and measure the differential binding affinities of monomeric and dimeric CF fragments to lipoprotein receptors to investigate the mode of canonical signal activation. We also present the cryoelectron tomography-solved dimeric structure of Reelin CF and support it using several other biophysical techniques. Our findings suggest that Reelin CF forms a covalent parallel dimer with some degree of flexibility between the two protein chains. As a result of this conformation, Reelin binds to lipoprotein receptors in a manner inaccessible to its monomeric form and is capable of stimulating canonical pathway signaling.

  View details for DOI 10.1016/j.str.2021.05.012

  View details for PubMedID 34089653

• Deep learning-based mixed-dimensional Gaussian mixture model for characterizing variability in cryo-EM. Nature methods Chen, M., Ludtke, S. J. 2021; 18 (8): 930-936

  Abstract

  Structural flexibility and/or dynamic interactions with other molecules is a critical aspect of protein function. Cryogenic electron microscopy (cryo-EM) provides direct visualization of individual macromolecules sampling different conformational and compositional states. While numerous methods are available for computational classification of discrete states, characterization of continuous conformational changes or large numbers of discrete state without human supervision remains challenging. Here we present e2gmm, a machine learning algorithm to determine a conformational landscape for proteins or complexes using a three-dimensional Gaussian mixture model mapped onto two-dimensional particle images in known orientations. Using a deep neural network architecture, e2gmm can automatically resolve the structural heterogeneity within the protein complex and map particles onto a small latent space describing conformational and compositional changes. This system presents a more intuitive and flexible representation than other manifold methods currently in use. We demonstrate this method on both simulated data and three biological systems to explore compositional and conformational changes at a range of scales. The software is distributed as part of EMAN2.

  View details for DOI 10.1038/s41592-021-01220-5

  View details for PubMedID 34326541

  View details for PubMedCentralID PMC8363932

• Preliminary Structural Elucidation of β-(1,3)-glucan Synthase from Candida glabrata Using Cryo-Electron Tomography. Journal of fungi (Basel, Switzerland) Jiménez-Ortigosa, C., Jiang, J., Chen, M., Kuang, X., Healey, K. R., Castellano, P., Boparai, N., Ludtke, S. J., Perlin, D. S., Dai, W. 2021; 7 (2)

  Abstract

  Echinocandin drugs have become a front-line therapy against Candida spp. infections due to the increased incidence of infections by species with elevated azole resistance, such as Candida glabrata. Echinocandins target the fungal-specific enzyme ß-(1,3)-glucan synthase (GS), which is located in the plasma membrane and catalyzes the biosynthesis of ß-(1,3)-glucan, the major component of the fungal cell wall. However, resistance to echinocandin drugs, which results from hotspot mutations in the catalytic subunits of GS, is an emerging problem. Little structural information on GS is currently available because, thus far, the GS enzyme complex has resisted homogenous purification, limiting our understanding of GS as a major biosynthetic apparatus for cell wall assembly and an important therapeutic drug target. Here, by applying cryo-electron tomography (cryo-ET) and subtomogram analysis, we provide a preliminary structure of the putative C. glabrata GS complex as clusters of hexamers, each subunit with two notable cytosolic domains, the N-terminal and central catalytic domains. This study lays the foundation for structural and functional studies of this elusive protein complex, which will provide insight into fungal cell wall synthesis and the development of more efficacious antifungal therapeutics.

  View details for DOI 10.3390/jof7020120

  View details for PubMedID 33562124

  View details for PubMedCentralID PMC7914498

• Structural Insights of Transcriptionally Active, Full-Length Androgen Receptor Coactivator Complexes. Molecular cell Yu, X., Yi, P., Hamilton, R. A., Shen, H., Chen, M., Foulds, C. E., Mancini, M. A., Ludtke, S. J., Wang, Z., O'Malley, B. W. 2020; 79 (5): 812-823.e4

  Abstract

  Steroid receptors activate gene transcription by recruiting coactivators to initiate transcription of their target genes. For most nuclear receptors, the ligand-dependent activation function domain-2 (AF-2) is a primary contributor to the nuclear receptor (NR) transcriptional activity. In contrast to other steroid receptors, such as ERα, the activation function of androgen receptor (AR) is largely dependent on its ligand-independent AF-1 located in its N-terminal domain (NTD). It remains unclear why AR utilizes a different AF domain from other receptors despite that NRs share similar domain organizations. Here, we present cryoelectron microscopy (cryo-EM) structures of DNA-bound full-length AR and its complex structure with key coactivators, SRC-3 and p300. AR dimerization follows a unique head-to-head and tail-to-tail manner. Unlike ERα, AR directly contacts a single SRC-3 and p300. The AR NTD is the primary site for coactivator recruitment. The structures provide a basis for understanding assembly of the AR:coactivator complex and its domain contributions for coactivator assembly and transcriptional regulation.

  View details for DOI 10.1016/j.molcel.2020.06.031

  View details for PubMedID 32668201

  View details for PubMedCentralID PMC7483370

• 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

• Structural and functional analyses of photosystem II in the marine diatom Phaeodactylum tricornutum. Proceedings of the National Academy of Sciences of the United States of America Levitan, O., Chen, M., Kuang, X., Cheong, K. Y., Jiang, J., Banal, M., Nambiar, N., Gorbunov, M. Y., Ludtke, S. J., Falkowski, P. G., Dai, W. 2019; 116 (35): 17316-17322

  Abstract

  A descendant of the red algal lineage, diatoms are unicellular eukaryotic algae characterized by thylakoid membranes that lack the spatial differentiation of stroma and grana stacks found in green algae and higher plants. While the photophysiology of diatoms has been studied extensively, very little is known about the spatial organization of the multimeric photosynthetic protein complexes within their thylakoid membranes. Here, using cryo-electron tomography, proteomics, and biophysical analyses, we elucidate the macromolecular composition, architecture, and spatial distribution of photosystem II complexes in diatom thylakoid membranes. Structural analyses reveal 2 distinct photosystem II populations: loose clusters of complexes associated with antenna proteins and compact 2D crystalline arrays of dimeric cores. Biophysical measurements reveal only 1 photosystem II functional absorption cross section, suggesting that only the former population is photosynthetically active. The tomographic data indicate that the arrays of photosystem II cores are physically separated from those associated with antenna proteins. We hypothesize that the islands of photosystem cores are repair stations, where photodamaged proteins can be replaced. Our results strongly imply convergent evolution between the red and the green photosynthetic lineages toward spatial segregation of dynamic, functional microdomains of photosystem II supercomplexes.

  View details for DOI 10.1073/pnas.1906726116

  View details for PubMedID 31409711

  View details for PubMedCentralID PMC6717305

• Stimulus-responsive self-assembly of protein-based fractals by computational design. Nature chemistry Hernández, N. E., Hansen, W. A., Zhu, D., Shea, M. E., Khalid, M., Manichev, V., Putnins, M., Chen, M., Dodge, A. G., Yang, L., Marrero-Berríos, I., Banal, M., Rechani, P., Gustafsson, T., Feldman, L. C., Lee, S. H., Wackett, L. P., Dai, W., Khare, S. D. 2019; 11 (7): 605-614

  Abstract

  Fractal topologies, which are statistically self-similar over multiple length scales, are pervasive in nature. The recurrence of patterns in fractal-shaped branched objects, such as trees, lungs and sponges, results in a high surface area to volume ratio, which provides key functional advantages including molecular trapping and exchange. Mimicking these topologies in designed protein-based assemblies could provide access to functional biomaterials. Here we describe a computational design approach for the reversible self-assembly of proteins into tunable supramolecular fractal-like topologies in response to phosphorylation. Guided by atomic-resolution models, we develop fusions of Src homology 2 (SH2) domain or a phosphorylatable SH2-binding peptide, respectively, to two symmetric, homo-oligomeric proteins. Mixing the two designed components resulted in a variety of dendritic, hyperbranched and sponge-like topologies that are phosphorylation-dependent and self-similar over three decades (~10 nm-10 μm) of length scale, in agreement with models from multiscale computational simulations. Designed assemblies perform efficient phosphorylation-dependent capture and release of cargo proteins.

  View details for DOI 10.1038/s41557-019-0277-y

  View details for PubMedID 31209296

• In situ structure and assembly of the multidrug efflux pump AcrAB-TolC. Nature communications Shi, X., Chen, M., Yu, Z., Bell, J. M., Wang, H., Forrester, I., Villarreal, H., Jakana, J., Du, D., Luisi, B. F., Ludtke, S. J., Wang, Z. 2019; 10 (1): 2635

  Abstract

  Multidrug efflux pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. In Gram-negative bacteria, these pumps form tripartite assemblies that span the cell envelope. However, the in situ structure and assembly mechanism of multidrug efflux pumps remain unknown. Here we report the in situ structure of the Escherichia coli AcrAB-TolC multidrug efflux pump obtained by electron cryo-tomography and subtomogram averaging. The fully assembled efflux pump is observed in a closed state under conditions of antibiotic challenge and in an open state in the presence of AcrB inhibitor. We also observe intermediate AcrAB complexes without TolC and discover that AcrA contacts the peptidoglycan layer of the periplasm. Our data point to a sequential assembly process in living bacteria, beginning with formation of the AcrAB subcomplex and suggest domains to target with efflux pump inhibitors.

  View details for DOI 10.1038/s41467-019-10512-6

  View details for PubMedID 31201302

  View details for PubMedCentralID PMC6570770

• A complete data processing workflow for cryo-ET and subtomogram averaging. Nature methods Chen, M. n., Bell, J. M., Shi, X. n., Sun, S. Y., Wang, Z. n., Ludtke, S. J. 2019

  Abstract

  Electron cryotomography is currently the only method capable of visualizing cells in three dimensions at nanometer resolutions. While modern instruments produce massive amounts of tomography data containing extremely rich structural information, data processing is very labor intensive and the results are often limited by the skills of the personnel rather than the data. We present an integrated workflow that covers the entire tomography data processing pipeline, from automated tilt series alignment to subnanometer resolution subtomogram averaging. Resolution enhancement is made possible through the use of per-particle per-tilt contrast transfer function correction and alignment. The workflow greatly reduces human bias, increases throughput and more closely approaches data-limited resolution for subtomogram averaging in both purified macromolecules and cells.

  View details for DOI 10.1038/s41592-019-0591-8

  View details for PubMedID 31611690

• Automation and assessment of de novo modeling with Pathwalking in near atomic resolution cryoEM density maps. Journal of structural biology Chen, M., Baker, M. L. 2018; 204 (3): 555-563

  Abstract

  With the rapidly growing number of macromolecular structures solved to near-atomic resolution using electron cryomicroscopy (cryoEM), map interpretation and model building directly from the density without the use of structural templates has become increasingly important. As part of the 2015/2016 Map and Model Challenge, we attempted to assess our latest de novo modeling tool, Pathwalking, in terms of performance and usability, as well as identify areas for future improvements. In total, we applied Pathwalking to six density maps between 3 and 4.5 Å resolution selected from the challenge data sets. In five of the six cases, Pathwalking was able to accurately determine the protein fold and in three of these cases, the final all atom model had less than 1.6 Å RMSD when compared to the known structure. Model building and refinement was nearly completely automated, used default parameters and took less than 30 min to complete a refined all atom model. A direct outgrowth of this work was a more streamlined automated command line Pathwalking utility, as well as a novel sequence assignment and optimization routine, which attempts to register sidechain density with expected side chain volume. In total, Pathwalking offers a nearly complete, robust and efficient method for constructing atomistic protein structures directly from a density map without the aid of a template.

  View details for DOI 10.1016/j.jsb.2018.09.005

  View details for PubMedID 30237066

• New software tools in EMAN2 inspired by EMDatabank map challenge. Journal of structural biology Bell, J. M., Chen, M., Durmaz, T., Fluty, A. C., Ludtke, S. J. 2018; 204 (2): 283-290

  Abstract

  EMAN2 is an extensible software suite with complete workflows for performing high-resolution single particle analysis, 2-D and 3-D heterogeneity analysis, and subtomogram averaging, among other tasks. Participation in the recent CryoEM Map Challenge sponsored by the EMDatabank led to a number of significant improvements to the single particle analysis process in EMAN2. A new convolutional neural network particle picker was developed, which dramatically improves particle picking accuracy for difficult data sets. A new particle quality metric capable of accurately identifying "bad" particles with a high degree of accuracy, no human input, and a negligible amount of additional computation, has been introduced, and this now serves as a replacement for earlier human-biased methods. The way 3-D single particle reconstructions are filtered has been altered to be more comparable to the filter applied in several other popular software packages, dramatically improving the appearance of sidechains in high-resolution structures. Finally, an option has been added to perform local resolution-based iterative filtration, resulting in local resolution improvements in many maps.

  View details for DOI 10.1016/j.jsb.2018.09.002

  View details for PubMedID 30189321

  View details for PubMedCentralID PMC6163079

• 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

• 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

• 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., Schmid, M. F., Chiu, W., He, C. Y. 2018; 115 (26): E5916–E5925

  View details for DOI 10.1073/pnas.1722618115

  View details for Web of Science ID 000436245000013

• 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

• 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

• 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): 983-985

  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

• De Novo modeling in cryo-EM density maps with Pathwalking. Journal of structural biology Chen, M., Baldwin, P. R., Ludtke, S. J., Baker, M. L. 2016; 196 (3): 289-298

  Abstract

  As electron cryo-microscopy (cryo-EM) can now frequently achieve near atomic resolution, accurate interpretation of these density maps in terms of atomistic detail has become paramount in deciphering macromolecular structure and function. However, there are few software tools for modeling protein structure from cryo-EM density maps in this resolution range. Here, we present an extension of our original Pathwalking protocol, which can automatically trace a protein backbone directly from a near-atomic resolution (3-6Å) density map. The original Pathwalking approach utilized a Traveling Salesman Problem solver for backbone tracing, but manual adjustment was still required during modeling. In the new version, human intervention is minimized and we provide a more robust approach for backbone modeling. This includes iterative secondary structure identification, termini detection and the ability to model multiple subunits without prior segmentation. Overall, the new Pathwalking procedure provides a more complete and robust tool for annotating protein structure function in near-atomic resolution density maps.

  View details for DOI 10.1016/j.jsb.2016.06.004

  View details for PubMedID 27436409

  View details for PubMedCentralID PMC5118137

• High resolution single particle refinement in EMAN2.1. Methods (San Diego, Calif.) Bell, J. M., Chen, M., Baldwin, P. R., Ludtke, S. J. 2016; 100: 25-34

  Abstract

  EMAN2.1 is a complete image processing suite for quantitative analysis of grayscale images, with a primary focus on transmission electron microscopy, with complete workflows for performing high resolution single particle reconstruction, 2-D and 3-D heterogeneity analysis, random conical tilt reconstruction and subtomogram averaging, among other tasks. In this manuscript we provide the first detailed description of the high resolution single particle analysis pipeline and the philosophy behind its approach to the reconstruction problem. High resolution refinement is a fully automated process, and involves an advanced set of heuristics to select optimal algorithms for each specific refinement task. A gold standard FSC is produced automatically as part of refinement, providing a robust resolution estimate for the final map, and this is used to optimally filter the final CTF phase and amplitude corrected structure. Additional methods are in-place to reduce model bias during refinement, and to permit cross-validation using other computational methods.

  View details for DOI 10.1016/j.ymeth.2016.02.018

  View details for PubMedID 26931650

  View details for PubMedCentralID PMC4848122

• 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