Classification of Single Particles from Human Cell Extract Reveals Distinct Structures

SUMMARY Multi-protein complexes are necessary for nearly all cellular processes, and understanding their structure is required for elucidating their function. Current high-resolution strategies in structural biology are effective but lag behind other fields (e.g., genomics and proteomics) due to their reliance on purified samples rather than heterogeneous mixtures. Here, we present a method combining single-particle analysis by electron microscopy with protein identification by mass spectrometry to structurally characterize macromolecular complexes from human cell extract. We identify HSP60 through two-dimensional classification and obtain three-dimensional structures of native proteasomes directly from ab initio classification of a heterogeneous mixture of protein complexes. In addition, we reveal an ~1-MDa-size structure of unknown composition and reference our proteomics data to suggest possible identities. Our study shows the power of using a shotgun approach to electron microscopy (shotgun EM) when coupled with mass spectrometry as a tool to uncover the structures of macromolecular machines.

[1]  Gary D Bader,et al.  Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry , 2002, Nature.

[2]  David N Mastronarde,et al.  Automated electron microscope tomography using robust prediction of specimen movements. , 2005, Journal of structural biology.

[3]  A. Horovitz,et al.  Conversion of the allosteric transition of GroEL from concerted to sequential by the single mutation Asp-155 → Ala , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[4]  Christopher Irving,et al.  Appion: an integrated, database-driven pipeline to facilitate EM image processing. , 2009, Journal of structural biology.

[5]  P. Bork,et al.  Capturing protein communities by structural proteomics in a thermophilic eukaryote , 2017, Molecular systems biology.

[6]  Anchi Cheng,et al.  Automated molecular microscopy: the new Leginon system. , 2005, Journal of structural biology.

[7]  Tom L. Blundell,et al.  DNA-PKcs structure suggests an allosteric mechanism modulating DNA double-strand break repair , 2017, Science.

[8]  W. Baumeister,et al.  Cryo-EM single particle analysis with the Volta phase plate , 2016, eLife.

[9]  Edward P. Morris,et al.  Cryo-EM reveals the conformation of a substrate analogue in the human 20S proteasome core , 2015, Nature Communications.

[10]  L. Foster,et al.  A high-throughput approach for measuring temporal changes in the interactome , 2012, Nature Methods.

[11]  M. Tinti,et al.  Global Membrane Protein Interactome Analysis using In vivo Crosslinking and Mass Spectrometry-based Protein Correlation Profiling* , 2016, Molecular & Cellular Proteomics.

[12]  Christian L. Müller,et al.  Identifying direct contacts between protein complex subunits from their conditional dependence in proteomics datasets , 2017, PLoS Comput. Biol..

[13]  W. Kühlbrandt.  The Resolution Revolution , 2014, Science.

[14]  Sean R. Collins,et al.  Global landscape of protein complexes in the yeast Saccharomyces cerevisiae , 2006, Nature.

[15]  P. Shannon,et al.  Cytoscape: a software environment for integrated models of biomolecular interaction networks. , 2003, Genome research.

[16]  Lennart Martens,et al.  SearchGUI: An open‐source graphical user interface for simultaneous OMSSA and X!Tandem searches , 2011, Proteomics.

[17]  Gaetano T. Montelione,et al.  The Protein Structure Initiative: achievements and visions for the future , 2012, F1000 biology reports.

[18]  D. Finley,et al.  Recognition and processing of ubiquitin-protein conjugates by the proteasome. , 2009, Annual review of biochemistry.

[19]  Edward L. Huttlin,et al.  The BioPlex Network: A Systematic Exploration of the Human Interactome , 2015, Cell.

[20]  J. Sacchettini,et al.  Crystal structure of the human 20S proteasome in complex with carfilzomib. , 2015, Structure.

[21]  M Radermacher,et al.  DoG Picker and TiltPicker: software tools to facilitate particle selection in single particle electron microscopy. , 2009, Journal of structural biology.

[22]  Hans-Werner Mewes,et al.  CORUM: the comprehensive resource of mammalian protein complexes , 2007, Nucleic Acids Res..

[23]  B. Böttcher,et al.  Precise mapping of subunits in multiprotein complexes by a versatile electron microscopy label , 2010, Nature Structural &Molecular Biology.

[24]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..

[25]  David J. Fleet,et al.  cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination , 2017, Nature Methods.

[26]  Greg W. Clark,et al.  Panorama of ancient metazoan macromolecular complexes , 2015, Nature.

[27]  Marco Y. Hein,et al.  A Human Interactome in Three Quantitative Dimensions Organized by Stoichiometries and Abundances , 2015, Cell.

[28]  Sjors H.W. Scheres,et al.  RELION: Implementation of a Bayesian approach to cryo-EM structure determination , 2012, Journal of structural biology.

[29]  R. Aebersold,et al.  Crosslinking and Mass Spectrometry: An Integrated Technology to Understand the Structure and Function of Molecular Machines. , 2016, Trends in biochemical sciences.

[30]  Eystein Oveland,et al.  PeptideShaker enables reanalysis of MS-derived proteomics data sets , 2015, Nature Biotechnology.

[31]  M. Gorenstein,et al.  Absolute Quantification of Proteins by LCMSE , 2006, Molecular & Cellular Proteomics.

[32]  N. Grigorieff,et al.  CTFFIND4: Fast and accurate defocus estimation from electron micrographs , 2015, bioRxiv.

[33]  M. Mirande.  The Aminoacyl-tRNA Synthetase Complex. , 2017, Sub-cellular biochemistry.

[34]  Andrei L. Turinsky,et al.  A Census of Human Soluble Protein Complexes , 2012, Cell.

[35]  W. Denk,et al.  Single-protein detection in crowded molecular environments in cryo-EM images , 2017, eLife.

[36]  Shoh M. Asano,et al.  A molecular census of 26S proteasomes in intact neurons , 2015, Science.

[37]  Claire D. McWhite,et al.  Integration of over 9,000 mass spectrometry experiments builds a global map of human protein complexes , 2017, Molecular systems biology.

[38]  Albert J R Heck,et al.  Interrogating the architecture of protein assemblies and protein interaction networks by cross-linking mass spectrometry. , 2015, Current opinion in structural biology.

[39]  P. Bork,et al.  Functional organization of the yeast proteome by systematic analysis of protein complexes , 2002, Nature.

[40]  Klaus Schulten,et al.  Structure of the human 26S proteasome at a resolution of 3.9 Å , 2016, Proceedings of the National Academy of Sciences.

[41]  E. Morris,et al.  Structure of the Human 26S Proteasome , 2008, Journal of Biological Chemistry.

[42]  Devin K. Schweppe,et al.  Architecture of the human interactome defines protein communities and disease networks , 2017, Nature.

[43]  Tian Xia,et al.  DeepPicker: a Deep Learning Approach for Fully Automated Particle Picking in Cryo-EM , 2016, Journal of structural biology.

[44]  Steven E Brenner,et al.  The Impact of Structural Genomics: Expectations and Outcomes , 2005, Science.

[45]  Gabriel C. Lander,et al.  Complete subunit architecture of the proteasome regulatory particle , 2011, Nature.

[46]  M. Mann,et al.  A generic strategy to analyze the spatial organization of multi-protein complexes by cross-linking and mass spectrometry. , 2000, Analytical chemistry.

[47]  F. Frolow,et al.  Crystal structure of the human mitochondrial chaperonin symmetrical football complex , 2015, Proceedings of the National Academy of Sciences.

[48]  A M Roseman,et al.  FindEM--a fast, efficient program for automatic selection of particles from electron micrographs. , 2004, Journal of structural biology.