UCLA

Biological nanomachines perform essential cellular functions through the coordinated organization of protein components across multiple structural scales. Understanding the structural principles governing these nanomachines is essential not only for elucidating their biological functions but also for enabling their engineering for biomedical and biotechnological applications. Cryogenic electron microscopy (cryoEM) and cryogenic electron tomography (cryoET) provide powerful tools for visualizing these systems from overall architectures to atomic-level interactions. This dissertation employs cryoEM and cryoET to investigate the structural basis of function in three distinct biological nanomachines: bacterial flagella, mitochondrial ATP synthase, and vault nanoparticles.First, the structural organization of the multi-flagellin flagellum from Shewanella oneidensis was characterized to investigate the molecular basis of bacterial motility. High-resolution cryoEM and cryoET analyses revealed the architectures of the flagellar filament, hook, and hook-filament junction, providing a comprehensive view of the structural elements responsible for torque transmission and propulsion. These findings advance the understanding of bacterial locomotion and establish structural principles governing the assembly and function of rotary motility machines. Second, vault nanoparticles were investigated as naturally occurring macromolecular nanomachines that can be engineered for therapeutic cargo delivery. CryoEM and cryoET studies of vault particles and engineered vault-interleukin-2 (IL-2) complexes revealed the molecular basis of cargo encapsulation through interactions between the INT domain and the major vault protein. Comparative analyses further demonstrated reduced structural flexibility upon cargo binding and uncovered substantial heterogeneity in cargo loading among individual vault particles. These findings provide a structural framework for understanding vault-mediated cargo encapsulation and inform future development of vault-based therapeutic delivery platforms. Lastly, ATP synthase complexes from mouse retina were characterized in their native membrane environment to investigate the structural architecture of mitochondrial energy-conversion machinery. High-resolution cryoEM structures of retinal ATP synthase provide a structural framework for understanding mitochondrial bioenergetics. Complementary cryoET analyses further characterized the organization of ATP synthase complexes within native mitochondrial membranes. These findings establish a structural foundation for investigating mitochondrial energy production and ATP synthase-associated diseases. Together, these studies provide insights into motility, energy conversion, and molecular encapsulation while demonstrating the power of cryoEM and cryoET for investigating biological nanomachines and guiding their engineering for biomedical applications.