UCLA

As the recent advances in cryogenic electron microscopy (cryoEM) have made it a tool of choice for determining biomolecular structures, it has been widely recognized that many complexes tend to have preferential orientation, aggregate, or disappear on cryoEM grids. However, the reasons for such misbehavior are not well understood, limiting the systematic approaches to resolve this problem. In this dissertation, we developed a theoretical formulation that explains these observations and rationalizes the use of surfactants in directly alleviating this phenomenon. We provided experimental evidence to demonstrate the effectiveness of various surfactants by visualizing the improved groEL particle distribution using cryogenic electron tomography (cryoET). We also conducted single particle analysis (SPA) on groEL to show that the surfactants do not adversely affect the protein structures at a near-atomic level. We further showed the effectiveness of surfactant on a more challenging membrane protein, CLC-1. In addition to testing the theoretical framework using a widely-test sample such as groEL, we also conducted a cryoET study of a more challenging zoonotic virus such as lymphocytic choriomeningitis virus (LCMV). The structures of individually isolated LCMV proteins or their fragments have been reported, but the architectural organization and the full-length structure of these proteins remain unknown. To showcase the benefits of using cryoET and sub-tomogram averaging (STA), we conducted a structural study on LCMV, unveiling the architectural organization of viral proteins of LCMV virion and resolving the full-length in situ structure of the glycoprotein complex (GPC) with its transmembrane domain intact. CryoET revealed a great variation in the number of polymerases within each virion, adding new perspectives to the infection mechanism. Finally, our structure proposes potential key residues that can be considered for structure-guided vaccine design against LCMV and arenavirus.