Classification of Single Particles from Human Cell Extract Reveals Distinct Structures
Eric J. Verbeke,
Anna L. Mallam,
Kevin Drew,
Edward M. Marcotte,
David W. Taylor
Affiliations
Eric J. Verbeke
Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712, USA; Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX 78712, USA; Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA
Anna L. Mallam
Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712, USA; Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX 78712, USA; Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA
Kevin Drew
Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712, USA; Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX 78712, USA; Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA
Edward M. Marcotte
Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712, USA; Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX 78712, USA; Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA; Corresponding author
David W. Taylor
Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712, USA; Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX 78712, USA; Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA; LIVESTRONG Cancer Institute, Dell Medical School, Austin, TX 78712, USA; Corresponding author
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. : Verbeke et al. demonstrate a shotgun approach to macromolecular structure determination by combining single-particle electron microscopy with mass spectrometry to reconstruct multiple three-dimensional models in a single experiment. This approach provides a method for investigating the structure and function of cellular machinery in parallel. Keywords: electron microscopy, structural biology, cellular fractionation, mass spectrometry, deep classification, heterogeneity analysis, protein complexes
