A Mid-Level 200kV Instrument for Single-Particle cryoEM

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Project Summary The goal of the California NanoSystems Institute (CNSI) at the University of California at Los Angeles (UCLA) is to enable cutting-edge nanobiology and biomedical research. As a key part of this goal, the Electron Imaging Center for Nanomachines (EICN) was established in 2007 to enable necessary advanced imaging techniques for visualizing and understanding the mechanisms of macromolecular machineries at the nanometer scale. Funding from a previous NIH S10 to EICN was instrumental to the purchase (2006) and installation (2007) of the world’s first working Titan Krios instrument, which contributed to the ‘cryoEM revolution’ of the last decade. This application seeks funding to purchase a Glacios, a mid-level 200 kV instrument with demonstrated high- resolution single-particle cryoEM imaging capability, to bridge the existing sets of entry-level and high-end Titan Krios instruments at the EICN at UCLA. The critical need for this instrument is justified as follows: First, the new instrument will meet the pressing need for single-particle cryoEM imaging in a broad range of federally funded biomedical research projects by 24 UCLA labs, including telomerase and spliceosomal nucleoprotein complexes; trans-membrane channels, transporters, bactericidal machineries and secretion systems; and complexes involved in neuro-degenerative diseases. Second, the existing 14-year old Titan Krios has developed age-related instability issues; additionally, 50% of its accessible time is reserved for an NIH U24 user consortium, while the other 50% is used to meet the quickly growing need of cryo-electron tomography (cryoET) users at UCLA, thanks to its high penetration power of 300 keV electrons and BioQuantum K3 Imaging filter. UCLA-based research would be greatly enhanced by the acquisition of this mid-level cryoEM for atomic structure determination by the single-particle cryoEM approach described in the application. The proposed instrument will enjoy strong institutional support thus ensuring lasting impact. High-resolution electron imaging has become an integral part of CNSI’s highly successful research resources and of the very strong structural biology research community at UCLA. The identification of tens of users with active federal funding shows that there is major interest across multiple departments/institutes among the colleges of natural sciences and engineering, as well as the UCLA medical school, which may greatly benefit from acquiring this mid-level cryoEM instrument with demonstrated high-resolution cryoEM capability. This instrument, together with existing micro-electron diffraction (microED), X-ray crystallography and NMR spectrometry, will provide a robust resource for faculty members who are eager to expand the scope of their current biomedical and biological research projects to include cryoEM. The new instrument will also meet a critical need for huge data sets for pushing the envelope of cryoEM for the cryoID method development. The diverse biological structures to be studied, and their highly varied architectures, offer a fertile data source for the scientific community for method development pursued by UCLA and the general electron imaging community at large.