The New York Structural Biology Center (NYSBC) is an instrumentation resource shared by nine New York academic institutions. NYSBC houses cryo-electron microscopes (cryo-EM), NMR spectrometers and X-ray facilities including a beam line at the NSLS synchrotron at Brookhaven National Laboratory. In this way, NYSBC provides high-end resources across the entire spectrum of structural biology. In order to maintain cutting-edge facilities in electron microscopy, we are seeking to upgrade the imaging detector on our top-end cryo-EM. Specifically, our JEOL 3200FSC electron microscope features a 300 keV field emission source with an in-column energy filter and a cartridge based specimen stage that provides excellent stability at either liquid nitrogen or liquid helium temperatures. At the time o purchase in 2005, the state of the art imaging device was a 4k x 4k CCD camera, which is currently used for a variety of projects involving single particles, tomography and 2D crystallography. However, this imaging technology produces a limitation to high resolution structure determination by our users. Rapid developments in direct electron detectors have occurred over the last several years and have been demonstrated to greatly outperform both these CCD cameras and film. Specifically the Gatan K2 Summit camera has been adopted by a number of laboratories and has produced dramatic results on biologically important macromolecules. These results have sparked a renewed interest in using cryo-EM for structure determination. By equipping our JEOL 3200FSC microscope with this camera, we aim to position NYSBC to provide these new opportunities to our diverse group of users. In particular, this upgrade will significantly improve image quality, thereby increasing the resolution for curren projects and facilitate new projects that were impractical with the existing CCD technology. Projects described in this application include tomography of unstained frozen specimens such as hepatitis C virus, microsporidia and bacteriophages, single particle analysis of complexes involved in cell adhesion, transcription, translation, nuclear and cytoplasmic transport, and electron crystallography of various membrane proteins. These projects will have a significant impact on our understanding of invasive pathogens and of essential cellular processes associated with cancer and with metabolic and neurodegenerative diseases. In addition to the PI, the NYSBC EM facility has three full-time, Ph.D. level staff who provide extensive hands-on experience in sample preparation, EM operation and computational image processing. The NYSBC EM facility has developed a robust system for maintenance and for managing the shared use of its instruments. Outreach to the community is accomplished through a semester-long course that is offered annually at NYSBC, though periodic advanced workshops and through a quarterly newsletter that is distributed to our expanding EM community. NYSBC provides a strong institutional commitment by supporting staff salaries, service contracts on all the electron microscopes and computational facilities required to take full advantage of this exciting new technology.
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