Modified Project Summary/Abstract Section We propose to establish a National Service Network Hub for Cryo-Electron Tomography to be hosted in the Department of Biochemistry at the University of Wisconsin, Madison. The Midwest Center for Cryo-Electron Tomography (MCCET) will support investigators by providing access to well-trained staff and state-of-the-art equipment for (1) routine and advanced cryo-electron tomography (cryo-ET) specimen preparation, data collection, and computation; and (2) hands-on, remote, and virtual training in cryo-ET specimen preparation, data collection, and data processing and validation. MCCET operations will build on the Cryo-EM Research Center (CEMRC) established in the Department of Biochemistry at the University of Wisconsin, Madison. The University of Wisconsin, Madison has a long history of institutional commitment to electron microscopy, as well as the advancement of electron microscopy in both the biological and materials sciences. The ancillary equipment and major instrumentation to be housed in the MCCET will include: cell culture equipment, a fluorescence light microscope and micropatterning system, glow discharge devices, carbon evaporator, automated plunge freezer, high pressure freezer, cryo-ultramicrotome, a cryo-focused ion beam-scanning electron microscope (cryo-FIB-SEM), and a 300 kV TEM. Dr. Elizabeth R. Wright, the contact PI, has over twenty-years of cryo-EM experience including the development of new cryo-EM technologies and workflows along with directing and managing (cryo-)electron microscopy research resources. Drs. Marisa Otegui, Robert Kirchdoerfer, and Tim Grant bring complementary expertise in sample preservation and imaging of complex tissues, small molecules and organelles, imaging and computational technology development, as well as facility management. A center manager, a computer scientist/systems administrator, a project manager, cryo- ET training instructors, a computer scientist, and postdoctoral research associates will manage MCCET day-today operations and support users through the course of their projects from initial access, sample handling and preparation, sample validation, cryo-ET data collection and processing, and cross-training. Hub coordination activities with the NIH, network centers, and users will be managed by the PI, project manager, center manager, and computer scientist/systems administrator. The Midwest Center for Cryo-Electron Tomography (MCCET) will maintain a culture of innovation and technology development in cryo-ET to facilitate improvements to cryo-ET sample preparation, throughput, validation, and imaging. The MCCET will be a user-experience focused resource that will provide access and training to researchers across many disciplines.