UCLA

Infections by viruses and flagellated protozoa lead to many human diseases, ranging from mild cold sores to devastating cancers. These pathogens infect billions of people worldwide and afflict wild and domestic animals, placing an enormous burden on global public health and economy. To resolve their in situ structures essential for infection, we have firstly established the validity of our approach of cryoET with sub-tomogram averaging using virus as a gold standard organism, then applied the same approach to investigate the structural details of flagellum in more complex protozoa Trypanosoma brucei.

In this thesis work, we firstly established the validity of our cryoET approach by obtaining in situ structures of the vesicular stomatitis virus glycoprotein G trimer in prefusion and postfusion conformations, which agree with the known crystal structures of purified G trimers in both conformations. Later for herpesvirus HCMV, we resolved 3D structures of gB trimers in two distinct conformations at up to 21 � resolution. We further captured the prefusion gB in complex with an “L”-shaped density attributed to the gH/gL complex. Our results deepen the knowledge of membrane fusion mechanism during HCMV infection.

How does nuclear egress complex (NEC)—the mediator of viral capsid budding into the cytoplasm—interacts with the viral capsid and how proper curvature of the coat is achieved to enable budding? We applied our cryoET approach to report that binding of a capsid protein, UL25, promotes the formation of a pentagonal rather than hexagonal NEC arrangement. Our results suggest that during nuclear budding, interactions between UL25 bound to the pentagonal capsid vertices and NEC introduce pentagonal insertions into the hexagonal NEC array to yield an NEC coat of the appropriate size and curvature, leading to productive budding and egress of UL25decorated capsids.

iii Furthermore, to understand structural foundations of eukaryotic flagella supporting motility and signaling necessary for infection, transmission, and pathogenesis, we then applied our cryoET approach to study T. brucei, a protozoan parasite in the Excavata lineage that causes African trypanosomiasis. From our resolved structure of its 96-nm axonemal repeats, we discovered several lineage-specific structures, including novel inter-doublet linkages and microtubule inner proteins. We also determined structures of the paraflagellar rod (PFR), PFR-axoneme connectors, and the axonemal central pair complex. Together, our findings fill the previously critical gap in understanding structural foundations of eukaryotic flagella, provide insights into flagellum-driven, non-planar helical motility of T. brucei and have broad implications ranging from cell motility and tensegrity in biology to engineering principles in bionics.