Classification of Single Particles from Human Cell Extract Reveals Distinct Structures

• Published in: Cell reports (Cambridge) Vol. 24; no. 1; pp. 259 - 268.e3
• Main Authors: Verbeke, Eric J., Mallam, Anna L., Drew, Kevin, Marcotte, Edward M., Taylor, David W.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 03.07.2018; Elsevier
• Subjects: Cell Extracts - chemistry; cellular fractionation; deep classification; electron microscopy; HEK293 Cells; heterogeneity analysis; Humans; Macromolecular Substances - metabolism; Macromolecular Substances - ultrastructure; mass spectrometry; Molecular Weight; Proteasome Endopeptidase Complex - metabolism; protein complexes; Protein Subunits - metabolism; structural biology; deep classification; heterogeneity analysis; electron microscopy; mass spectrometry; structural biology; protein complexes; cellular fractionation
• ISSN: 2211-1247; 2211-1247
• DOI: 10.1016/j.celrep.2018.06.022

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. [Display omitted] •Whole-cell extract can be fractionated and visualized using electron microscopy•Multiple 3D structures can be recovered from fractionated cell extract•Mass spectrometry data can inform on the identity of the resulting 3D structures•Using this method, proteasomes in two different biological states are observed 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.