CryoEM-Enabled Structural Studies Of Challenging Protein Complexes At Atomic Resolution

Structures are key to understanding the molecular mechanism of basic biological processes.Conventional structural determination approaches such as X-ray crystallography and nuclear magnetic resonance(NMR)have leaded to thousands of protein structures,greatly contributing to our knowledge of how cells perform important biological tasks.However,as X-ray crystallography requires protein crystals,it has dampened the structural studies of important proteins especially protein complexes and membrane proteins which fail to express well in recombinant systems or refuse to crystalize.As with NMR,it needs large amounts of pure samples and is only suitable for small proteins.Recently cryoEM has evolved as a routine approach for structural determination of proteins at atomic resolution,featured by minimum requirement of sample concentration or purity.Here I present two successful applications of cryoEM to challenging protein complexes or organisms which resist conventional structural characterization approaches.Frist,we take advantages of cryoEM single particle analysis to elucidate the molecular mechanism underlying the specific recognition of CENP-A nucleosome(NCP)by CENP-LN complexes,a critical step for the assembly of functional kinetochore to enable accurate chromosome segregation in mitosis.Harmessing the advantages of cryoEM single particle analysis,we managed to overcome the heterogeneity of CENP-LN lCENP-A NCP sample which refused X-ray crystallography approach,and got its cryoEM structure with an overall resolution of 5.8 A.We found that CENP-N interacts both with CENP-A LG loop and nucleosome DNA to specifically recognize CENP-A containing NCP.Furthermore,we present a bottom-up endogenous structural proteomics approach for challenging organisms,which enables us to get multiple atomic resolution cryoEM density maps from samples containing various endogeneous protein complexes.In our approach,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 are identified using cryoID,a program we developed to identify proteins in ab initio cryoEM maps from thousands of candidate sequences.As a proof of principle we applied our approach to the challenging malaria parasite Plasmodium falciparum and obtained multiple protein complexes structures at atomic resolution.Taken together,our results show the great power of cryoEM for the structural studies of challenging protein complexes at atomic resolution of previously intractable biological systems.