PROJECT SUMMARY Recent technological advances in cryogenic-electron microscopy (cryo-EM) have heralded a “resolution revolution” that has made it possible for high-resolution structures to be determined for a variety of targets that had eluded other structural techniques. Despite these advances, cryo-EM grid preparation remains a formidable roadblock to successful structure determination in many cases. Indeed, despite improvements in electron optics, camera technology, and image analysis, and a general drive towards automation, cryo-EM grid preparation remains a laborious and unpredictable process that requires extensive manual user input for optimization. The University of California, San Diego (UCSD) has established a world-class cryo-EM facility with state-of-the-art electron microscopes and staff, yet numerous projects across campus remain stalled due to an inability to prepare high-quality cryo-EM grids from purified specimens. To address this bottleneck in the cryo-EM workflow at UCSD, we propose to purchase an SPT Labtech Chameleon sample vitrification instrument. This instrument represents the first major innovation in cryo-EM grid preparation in over 30 years, with an entirely novel approach to sample application that yields more reproducible cryo-EM grids, improves particle quality and distribution, and generally increases the likelihood of high-resolution structure determination. The instrument will be centrally housed and operated within the recently renovated UCSD Cryo-EM Facility and will be available to all trained users at UCSD, surrounding institutions in the La Jolla “Mesa” area, and across California. For users at UCSD, this instrument will catalyze progress in a broad range of NIH-funded, biomedically-important projects spanning neurodegeneration, virology, membrane trafficking, DNA replication and repair, immunology, and protein biogenesis and folding.