This application requests funds to purchase an FEI Falcon 2 direct electron detecting (DED) camera for use with the Polara microscope of the cryo-electron microscopy research core at the University of Pittsburgh. This resource houses a cluster of microscopes (2 cryo, one non-cryo) and serves all members of the University of Pittsburgh who wish to apply single particle imaging and cryo-tomography in their research. As there are no other cryo-EM systems in the local area, or indeed in Western Pennsylvania, we collaborate with NIH-funded investigators across multiple campuses. Currently all our microscopes are equipped with both film cameras and CCD detectors. While film is an excellent recording medium with high sensitivity, resolution and area, the water vapor it introduces rapidly contaminates frozen grids, degrading image quality. This coupled with slow throughput and manual cassette exchange precludes film from use with automated data collection procedures that are essential for acquiring the large datasets needed for working at the limits of resolution or for batch cryo-tomography. CCD cameras are compatible with automation and do collect images of sufficient quality and quantity for standard cryoEM investigations. However, the core CCD technology absolutely limits resolution by smearing (i.e., de-localizing) imaging electrons when converting them to detectable photons. The introduction of DED cameras has completely revolutionized this field by coupling automated imaging with both very high resolution and outstanding signal/noise (i.e., sensitivity and contrast). The preliminary DED data shown in this application demonstrate near-atomic resolution structures for two 600 -diameter virus capsids where large side-chains are visualized in addition to helix chirality and ss-strand separation. Additional cutting edge capabilities embodied in this technology will also be necessary for ultimately achieving atomic resolution, such as sub-frame alignment to correct for drift and beam-induced movement. The high-speed image read-out also improves efficiency by increasing data quantity since it negates time lost to both film exchange and outgassing (hours) as well as the CCD image read-out delay (20 secs/frame) that currently accounts for ~2/3 of multi-day automated acquisition runs. Two recent publications presented atomic resolution structures derived from DED cameras mounted on Polara cryo-microscopes at UCSF and at the MPI-Frankfurt that are identical to ours, demonstrating the suitability of our equipment for hosting a DED camera. In summary, acquisition of this DED instrument will have immeasurable impact in our research areas and will completely change the fabric of cryoEM at our institution by greatly extending our reach in structure analysis and understanding how protein complexes function.
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