Classification of Single Particles from Human Cell Extract Reveals Distinct Structures

Cell Rep. 2018 Jul 3;24(1):259-268.e3. doi: 10.1016/j.celrep.2018.06.022.

Abstract

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.

Keywords: cellular fractionation; deep classification; electron microscopy; heterogeneity analysis; mass spectrometry; protein complexes; structural biology.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Cell Extracts / chemistry*
  • HEK293 Cells
  • Humans
  • Macromolecular Substances / metabolism*
  • Macromolecular Substances / ultrastructure
  • Molecular Weight
  • Proteasome Endopeptidase Complex / metabolism
  • Protein Subunits / metabolism

Substances

  • Cell Extracts
  • Macromolecular Substances
  • Protein Subunits
  • Proteasome Endopeptidase Complex