Classification of Single Particles from Human Cell Extract Reveals Distinct Structures

摘要

Multi-protein complexes are necessary for nearly allcellular processes, and understanding their structureis required for elucidating their function. Currenthigh-resolution strategies in structural biology areeffective but lag behind other fields (e.g., genomicsand proteomics) due to their reliance on purifiedsamples rather than heterogeneous mixtures. Here,we present a method combining single-particleanalysis by electron microscopy with protein identificationby mass spectrometry to structurally characterizemacromolecular complexes from human cellextract. We identify HSP60 through two-dimensionalclassification and obtain three-dimensional structuresof native proteasomes directly from ab initioclassification of a heterogeneous mixture of proteincomplexes. In addition, we reveal an 1-MDa-sizestructure of unknown composition and referenceour proteomics data to suggest possible identities.Our study shows the power of using a shotgunapproach to electron microscopy (shotgun EM)when coupled with mass spectrometry as a tool touncover the structures of macromolecular machines.