Project Summary/Abstract We propose to acquire a state-of-the-art 200 kV Glacios Cryo transmission electron microscope at Case Western Reserve University School of Medicine (CWRU SOM) for structural studies of biological macromolecular assemblies. Cryo-electron microscopy (Cryo-EM) has emerged as an unparalleled tool for high-resolution structure determination of macromolecules ranging from sizes as low as 40 kDa and as high as several MDa. The technology allows for imaging samples in solution, native membrane bilayers, or even in their physiological cellular environment. This new microscope will extend the capabilities of the Cryo-EM Core Facility at CWRU SOM in single-particle Cryo-EM and Cryo- electron tomography (Cryo-ET). Currently, the Cryo-EM needs of the research community in the Greater Cleveland area are supported by three transmission electron microscopes: Tecnai T12, Tecnai TF20, and most recently added Krios G3i. The Cryo-EM Core provides access to users from CWRU, its regional affiliate institutions, and other Universities in Northeast Ohio. The major technological advancements in Cryo-EM have made this methodology easier to adopt for diverse biological systems. Combined with NIH-supported Cryo-EM training programs and increased institutional support for collaborative initiatives, these developments have led to a major increase in the demand for pursing Cryo-EM. While our high- resolution instrumentation is state-of-the-art, our productivity is severely affected by our aging screening microscopes, which, although maintained under continued service contracts, have increasingly frequent and prolonged periods of repair. The proposed Glacios microscope will meet the substantial needs of seven major users and eight minor users. This microscope is a major advancement over our current 200 kV microscope featuring better electron optics, column stability, and more importantly is equipped with a larger capacity sample- handling robot that is directly compatible with the Krios G3i. Advanced features of the proposed instrument that are critical to the success of the proposed experiments include: a) significantly improved sensitivity for smaller- sized samples with poor contrast; b) enhanced throughput for Cryo-EM/Cryo-ET sample screening, optimization, and high-resolution data collection to cater to the growing needs of the structural biologists; c) ease of training, and better integration within the existing Cryo-EM workflow. The NIH-funded research projects that will directly benefit from this instrumentation include the structural studies of a number of clinically relevant targets such as ion channels, membrane signalling receptors, prion proteins, immune receptors, and viruses. Overall, the acquisition of the new microscope will expand the scope of biomedical research and further strengthen the multidisciplinary collaborative interactions within our scientific community here in the Greater Cleveland area.