PROJECT SUMMARY Despite modern advances in structural biology, structures of many biomedically relevant macromolecular assemblies remain out of reach or lack atomic resolution detail. In addition, the process of determining entirely novel structures remains laborious. Large crystals required for conventional crystallography experiments are a challenge to grow, and determination of structures from small or imperfect crystals by x-ray crystallography remains limited. Cryo- electron microscopy (cryo-EM) methods promise to bring new life to high-throughput approaches in macromolecular structure determination. The cryo-EM revolution has brought with it new high-resolution methods including micro electron diffraction (MicroED). MicroED exploits the strong interaction between electrons and nano-scale three-dimensional crystals by leveraging emerging cryo-EM instrumentation against established crystallographic knowledge. My group has helped achieve milestone discoveries in MicroED and determined entirely new protein structures from crystals and crystal fragments smaller than the wavelength of visible light, at atomic resolution. These technological advances, coupled with the greater availability of advanced cryoEM instruments, present an opportunity for further improvement of high- throughput structure determination. The development new and more efficient approaches to structure determination by MicroED could open new avenues for comprehensive exploration of complex macromolecular structures that remain out of reach for standard methods. These systems include macromolecular complexes that grow small, fragile, or imperfect crystals. The biomedical problems associated with these types of assemblies are varied and could broadly impact biomedicine, both through the basic interpretation of disease and as therapeutic platforms. Specifically, we aim to target infectious and/or toxic filamentous nanoassemblies associated with amyloid disease. Through our efforts in determining these challenging structures, we find inspiration to guide the improvement and development of cryoEM techniques, particularly MicroED. With this goal in mind, my group will take on the development of new high-throughput methods for crystallographic structure determination while obtaining structures of pressing biomedical targets at truly atomic resolution.