With the completion of a number of genome sequences and the resultant catalysis of basic biomedical research there is a realization that the individual proteins of the cell function for the most part as components of larger macromolecular assemblies- the molecular machines of the cell. These machines are likely to be conformationally and compositionally dynamic, as well as being present in comparatively low numbers, making them extremely challenging objects for study by x-ray crystallographic or NMR approaches. They are however ideal for EM structure determination. This approach allows calculation of moderate resolution (10-30A) 3D maps that can provide unique structural and mechanistic insights into the action of the machines. Insights into the structure and function of the ribosome and various families of motor proteins provide two important examples of the power of the technique. Although the methodology of molecular microscopy has enormous potential, it is time consuming and labor intensive. The goal of the proposed application is to transform EM structure determination into a high through put mainstream methodology. Success in this endeavor will not only greatly facilitate the process of molecular microscopy but also will expand the scope of accessible problems and push experimental frontiers by making possible investigations that are presently deemed too high risk because of the enormous effort involved. We thus propose to establish a National Resource for Automated Molecular Microscopy based at The Scripps Research Institute. Core technology projects will address automation and high throughput methods for specimen labeling, specimen handling, image acquisition, data processing and data information integration. Our unique contribution will be to develop, test and apply technology aimed towards completely automating the processes involved in solving macromolecular structure using cryo-electron microscopy (cryoEM). Our overall goal will be to establish a resource that will serve both as a center for high-throughput molecular imaging as well as for transferring this technology to the research community.
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