UCLA

The last two decades have seen revolutionary progress in the field of cryo electron microscopy, granting views of previously unresolved biological structures utilizing both electron diffraction and electron imaging. Microcrystal electron diffraction (MicroED) has become applicable to protein, peptide, and small molecule structures while cryo-electron microscopy (cryo-EM) has allowed for high-resolution structure determination of proteins and macromolecular complexes outside the confines of crystallographic restraints. The use of MicroED to interrogate a wider variety of structures has revealed important molecular insights in the worlds of biology and chemistry but has also revealed some of the shortcomings of the method that could be limiting to novel structure determination. Namely, there remains a limited selection of phasing methodologies for electron diffraction data: molecular replacement, requiring a highly similar structural model, or direct methods, requiring an atomic resolution dataset. The experiments presented in chapters 2 and 3 of this thesis explore the implementation of fragment-based phasing methodologies to electron diffraction data to overcome these limitations and demonstrate their applicability to protein and amyloid peptide structures. Electron microscopy has been particularly important in the study of amyloid proteins. The defining characteristic of amyloid proteins is the formation long, unbranched fibrillar aggregates which can be disease-associated, as is observed in many neurodegenerative diseases, or functional within the context of cell. Short peptide segments of these proteins which recapitulate the underlying cross-β structure of the fibrils have been lucrative targets for both X-ray crystallography and MicroED due to the small size of crystals they form. Meanwhile, improvements in cryo-EM resolution and data processing have permitted the first high-resolution views of full-length amyloid fibril structures. The fourth chapter of this thesis takes advantage of these advancements to elucidate the structure of the amyloid fibril form of human leukocyte cell-derived chemotaxin-2 (LECT2) by cryo-EM. ALECT2, the systemic amyloid disease associated with LECT2 aggregation, is clinically well characterized but poorly understood molecularly. While predictions of the aggregation-prone segments which contribute to amyloid formation have been made, a lack of structural information has limited the rational design of targeting agents and therapeutics. The work presented here offers a first step in uncovering the molecular mechanisms behind LECT2 aggregation and their role in ALECT2 disease progression.