Improvement of High resolution data collection in macromolecular electron cryo-microscopy

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Electron cryo-microscopy and image analysis have become useful tools for determining 3-dimensional structures of macromolecular assemblies at near atomic resolution. Our laboratory is uniquely equipped with a 400 kV electron microscope funded by the National Center for Research Resources of NIH as a national facility. To complement our NIH support, this proposal is aimed at improving the specimen preparation procedure,, cryo-specimen holder performance and data collection on our 400 kV electron cryo- microscope. The first half of this proposed technology development will be carried out in collaboration with Gatan Inc. to build a carbon cryo-coater for vacuum deposit of a thin carbon film onto frozen, hydrated specimens, and high resolution cryo-specimen holder for the JEOL 400 kV electron microscope. The performance of this equipment will be evaluated with a variety of biological specimens under investigation in our Resources Center. The final version of this equipment will become standard products of Gatan. The second half of this proposal is to develop software for capture and analysis of electron diffraction patterns and images of biological specimens with our newly acquired 1024x1024 slow scan charge-coupled device (CCD) camera. This software will be made available to the research community. These enchanced technologies will be used for several collaborative and user projects in our NIH supported Resource Center.