Bottom-up Structural Proteomics: Cryo-EM of Protein Complexes Enriched from the Cellular Milieu

Abstract

X-ray crystallography and recombinant protein production have enabled an exponential increase in atomic structures, but often require non-native constructs involving mutations or truncations, and are challenged by membrane proteins and large multi-component complexes. We present here a bottom-up endogenous structural proteomics approach whereby near-atomic resolution cryoEM maps are reconstructed ab initio from unidentified protein complexes enriched directly from the endogenous cellular milieu, followed by identification and atomic modeling of the proteins. The proteins in each complex are identified using cryoID, a program we developed to identify proteins in ab initio cryoEM maps. As a proof of principle, we applied this approach to the malaria parasite Plasmodium falciparum, an organism that has resisted traditional structural biology approaches, to obtain atomic models of multiple protein complexes implicated in intraerythrocytic survival of the parasite. Our approach opens the door to atomic structures of previously intractable biological systems.