PROJECT SUMMARY We propose to acquire a Thermo Scientific Glacios, a 200kV cryo-transmission electron microscope, to advance our biological investigation using the growing technologies of cryo-EM. The Glacios will be a part of the Cryo-EM Facility, a shared resource of the Center for Structural Biology. Our investigators have established records of using single particle analysis and discovering novel biological molecular mechanisms at the atomic level. The addition of the Glacios will be essential for our investigators to further advance their research beyond what is currently possible using our existing instruments. Specifically, the Glacios will be essential for: (1) structural analyses of low molecular weight proteins utilizing 200kV acceleration voltage, (2) the MicroED approach that uses electron diffraction of micro-crystals, (3) investigation of macromolecular complexes in situ using cryo- electron tomography, and (4) specimen optimization to advance our efficiency to resolve at high- resolution fine conformational varieties and small molecules, such as ligands, drugs, and ions. The technology provided by the Glacios is essential for breaking new ground on the NIH-funded research projects of the major users, which include studies of synaptic transmission and plasticity (Nakagawa), in situ synapse architecture and synaptic vesicle fusion (Zhou), Clostridioides difficile toxin pathology (Lacy), nuclear mRNA transport (Ren), DNA replication and repair (Eichmann), calcium signaling (Karakas), in situ viral assembly and architectures (Wan), vesicle and membrane trafficking (Jackson), and bacterial signaling (Iverson). Additionally, 11 minor users plan to exploit the Glacios to investigate critical biological questions in membrane protein function, inflammatory signaling, genome maintenance, bacteria-host interactions, ion channel mediated signaling, lipid signaling and biosynthesis, and molecular motors. The outputs of the research projects that will use the Glacios will substantially advance our basic understanding on the mechanisms of human physiology, pathology, and disease therapeutics. The Glacios will initially be used by structural biologists and cell biologists, with the userbase predicted to extend into the fields of chemistry and bioengineering who have related scientific interests. In fact, the Glacios will be housed in the Engineering and Science Building, which is designed to nurture collaborations between disciplines via a three-floor, integrative, and collaborative laboratory space linking the School of Medicine, College of Arts and Sciences, and the School of Engineering.