UCLA

As we seek to elucidate the functions and mechanisms of biological constructs, there is a growing need to learn about their structures in their native contexts as members of their molecular societies. Conventional methods of structural biology entail purification of recombinant proteins, which isolates them from their interactome and spatial localization. Thus, minimizing sample manipulation is vital for discovering biological interactions. Unique among the structural biology methods, cryogenic electron microscopy (cryoEM) is able to generate data from impure, heterogeneous samples. By combining old-fashioned endogenous methods with recent advancements in the throughput of cryoEM data collection and computation, we can perform purification in silico, allowing for high resolution structural determination of proteins while retaining their native binding partners. We apply this endogenous cryoEM approach to determine the structures of two critical metabolic complexes, the icosahedral pyruvate dehydrogenase complex (PDC) and the cubic 2-oxoglutarate dehydrogenase complex (OGDC), from mitochondrial lysates of bovine kidneys. By minimizing disruption of the sample, we identify heterogeneous arrangements of E1 and E3 peripheral subunits bound to their central E2 cores. Additionally, the substrate-shuttling lipoyl moiety of E2 is found within the E2 active site. Direct comparison of PDC and OGDC from the same organism allows us to identify critical interaction interfaces for the prevention of heterologous binding and determination of geometry despite a near-identical monomeric E2 fold. Some systems do not have the luxury of containing multiple copies for averaging by single particle cryoEM. Instead, cryogenic electron tomography (cryoET) enables imaging of unique subjects within their fully intact environment. Here, we report structures and observations of intact cancer-causing herpesviruses, Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV), and their pleiomorphic tegument and envelope using cryoET. Within their teguments, we observe strand-like features that suggest a means of viral assembly. We also show that herpesvirus fusogen proteins assemble into triplet clusters to promote host membrane fusion. Our results provide new insights into assembly of metabolic complexes and herpesviruses. Together, they highlight wide applicability of cryoEM and cryoET for understanding large complexes in context of their native systems.