A. Project Summary This proposal is for the purchase of a user-friendly ThermoScientific Tundra transmission cryo-electron microscope (cryo-EM) and an accompanying, highly automated Vitrobot V cryo-plunger, to advance biomedical research on the Boston University Charles River and Medical School campuses. This novel microscope has been developed to democratize the cryo-EM ‘resolution revolution’. Currently, there is no cryo-EM at Boston University that is capable of high-resolution structure determination. The existing lower- resolution TF20 is over 20 years old, can only be used by experts with extensive training, and is incapable of generating data suitable to solve state-of-the-art structures that are of interest to an established group of 25 biomedical researchers. The onsite installation of the proposed Tundra cryo-EM will be a game changer for research programs of the investigators. The new cryo-EM has a ‘traffic light’ system for setup, data collection, and troubleshooting, making entry-level work user-friendly. If funded, the automation and relative simplicity of use of the Tundra cryo-EM and the accompanying Vitrobot system will enable both cryo-EM experts and non-experts to determine structures of macromolecular assemblies in a timely-manner at a reasonable cost. Structure determination by our user group will advance the NIH-funded projects of a broad range of biomedical scientists, many of whom have not had previous access to cryo-EM, allowing them to address more in-depth research questions by using detailed structural information to elucidate function. We are enthusiastic about adding this capability for Virologists, Enzymologists, Chemists, Molecular Biologists, Molecular Physiologists, Neuroscientists, and other researchers to visualize their samples and determine structures of biomedically relevant macromolecules at a resolution that may reach 3.5 to 4 Å. The associated Vitrobot plunge-freezing robot and Autogrid cassette mounting system on the Tundra would allow users to prepare and screen grids for local use and, if needed, for high-resolution data collection at National Cryo-EM Centers or fee-for-service Cryo-EM facilities. Importantly, the requested instruments will streamline sample preparation and increase user productivity. The solved 3D structures of biological complexes can provide important insights on how they function; thus, data generated from the proposed Tundra cryo-EM will advance our understanding of the mechanisms of biological systems with unparalleled precision and provide insights into addressing molecular dysfunctions that correlate with disease states.

B. Project Narrative This proposal is for the purchase of a newly designed, user-friendly, Tundra cryo-electron microscope and an accompanying, highly automated cryo-plunger for sample preparation. This novel microscope has been developed with a `traffic light' system for setup, data collection, and troubleshooting, where green is `go', yellow is `caution' and red is `stop'. The microscope's ease of use will make it possible for our 25 research groups comprised of both experts and non-experts to determine biomedically important structures at near- atomic resolution, and thereby elucidate the molecular basis of diseases that range from viral and bacterial infections to Alzheimer's, cardiovascular disease and beyond.