Our objectives are to determine the three-dimensional (3D) crystal structures of macromolecules at near atomic resolution and the 3-D structures of non-crystalline macromolecular assemblies at intermediate resolution, by a combination of electron microscopy and computer processing methods. The present proposal is a resource related project. Requests include the acquisition of a medium high voltage (300 kV) electron microscope with appropriate accessories and an operational fund to develop the technology for the above aims. Technical development will be focused on specimen preparation, 3-D data collection at low dose and low temperature conditions and implementation of computer programs for 3-D reconstruction. The developed technology will be immediately applied to the high resolution crystal structure determination of crotoxin complex, T4 DNA helix destabilizing protein, fungus lysozyme and Fc fragments; and to the intermediate resolution structure determination of muscle Z disc and flagella axoneme. Collaborative projects will be established with investigators outside our research groups after the midpoint of the grant period. We plan to complete a thorough analysis of the potential as well as limitations of this technology in its general applicability to a variety of biological systems. We expect to present a final recommendation on the feasibility and desirability of establishing a national shared facility of this technology at the end of this grant period.
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