National Institutes of Health/National Center for Research Resources (NIH/NCRR)
R01GM071940
米国
National Institutes of Health/National Center for Research Resources (NIH/NCRR)
AI094386
米国
National Institutes of Health/National Center for Research Resources (NIH/NCRR)
DE025567
米国
National Institutes of Health/National Center for Research Resources (NIH/NCRR)
S10RR23057
米国
National Institutes of Health/National Center for Research Resources (NIH/NCRR)
S10OD018111
米国
National Institutes of Health/National Center for Research Resources (NIH/NCRR)
U24GM116792
米国
National Science Foundation (NSF, United States)
DBI-1338135
米国
National Science Foundation (NSF, United States)
DMR-1548924
米国
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
T32 AI007323
米国
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)
K99/R00 HL133453
米国
引用
ジャーナル: Nat Methods / 年: 2020 タイトル: Bottom-up structural proteomics: cryoEM of protein complexes enriched from the cellular milieu. 著者: Chi-Min Ho / Xiaorun Li / Mason Lai / Thomas C Terwilliger / Josh R Beck / James Wohlschlegel / Daniel E Goldberg / Anthony W P Fitzpatrick / Z Hong Zhou / 要旨: X-ray crystallography often requires non-native constructs involving mutations or truncations, and is challenged by membrane proteins and large multicomponent complexes. We present here a bottom-up ...X-ray crystallography often requires non-native constructs involving mutations or truncations, and is challenged by membrane proteins and large multicomponent complexes. We present here a bottom-up endogenous structural proteomics approach whereby near-atomic-resolution cryo electron microscopy (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-causing parasite Plasmodium falciparum, an organism that has resisted conventional structural-biology approaches, to obtain atomic models of multiple protein complexes implicated in intraerythrocytic survival of the parasite. Our approach is broadly applicable for determining structures of undiscovered protein complexes enriched directly from endogenous sources.