We seek funds to establish a center for advanced molecular microscopy. The requested instrumentation includes a Philips CM200T FEG; a Philips CM120-cryo; a Gatan cryo-transfer system, anticontaminator, and dry-pumping station for each of the microscopes; a Gatan 1k x 1k slow-scan CCD and a Gatan Imaging Filter. The choice of Philips instruments is based on ease of use and known quality of the instruments, and the reliability and quality of the service from this company. The new microscopes and cryo-accessories will replace existing 8-year-old equipment which, based on preliminary data on our specimens collected on a CM200T-FEG, is not adequate in terms of performance or resolution for obtaining the data we need to proceed to the next level of structural understanding of our specimens. Based on tests carried out over the last 6 months, the new instruments will dramatically improve both the quality and the quantity of data that can be collected. The field emission electron gun (FEG) provides a more coherent electron beam for imaging. Consequently, the image contrast transfer function remains strong to much higher resolution than on conventional images, thus allowing higher resolution data to be extracted from the images. Our preliminary data indicate that at both moderate (20-40 and high (3.5-10 resolution, the CM200T-FEG provides very significant - sometimes dramatic - improvements in the resolution obtained in images of our specimens. Both microscopes incorporate the new Philips "Compu-Stage" - a much-improved, computercontrolled goniometer. The design of the new goniomenter (there are no contacts with the low temperature part of the cold stage) results in much greater specimen stability. The cold stage equilibrates very rapidly after insertion and after stage translations. We estimate that these advantages will speed up data collection by a factor of 5-10. The research of the user group focuses on 3-D molecular microscopy: determination of the near-atomic, s econdary, tertiary and quaternary structure of molecules and macromolecular assemblies by cryo-electron microscopy and image analysis. The common goal of the user group is to calculate 3-D structures of biological molecules and macromolecular complexes to determine how they work. In all cases, higher resolution than can currently be achieved with our present instrumentation is essential for progress. The biological specimens under investigation include acto-myosin, ncd-microtubule and kinesin-microtubule complexes (Milligan), membrane proteins and ion channels (Milligan, Mitra, Unwin, Yeager), plant and animal viruses (Beachy, Johnson, Yeager), and synthetic organic nanotubes (Ghadiri). Training is an integral part of the activities. Postdoctoral fellows and graduate students will receive advanced theoretical and practical training in high resolution cryo-electron microscopy (imaging and electron diffraction), image analysis, high resolution structure determination and structure interpretation. The proposed center for advanced molecular microscopy will incorporate the following technologies: cryo-electron microscopy, <200kV imaging and electron diffraction, a field emission electron source, analog and digital image acquisition, and zero-energy-loss imaging and electron diffraction. Together with the computational and visualization capabilities for image analysis and structure determination that already exist within the user group, the requested instrumentation will create a unique research and training facility which will have a major impact on the regional, national and international research infrastructure.

National Science Foundation (NSF)
Division of Biological Infrastructure (DBI)
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Lee C. Makowski
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The Scripps Research Institute
La Jolla
United States
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