We propose to assemble a technology development and structure determination pipeline for high throughput structural biology. Our emphasis will be on sample preparation for diffraction study. Our technology development efforts will center on (1) the expression and purification of small protein - protein complexes from yeast;(2) the expression and purification of transmembrane proteins from yeast;(3) the identification, expression and purification of antibodies that target transmembrane proteins from yeast, as aids to their crystallization;(4) the crystallization of protein complexes, transmembrane proteins, transmembrane protein - antibody complexes, and soluble proteins;and (5) the cryopreservation of protein crystals. All of the technology development efforts will be at, or eventually geared to, high throughput. Our structure determination pipeline will run at medium throughput, and will take projects emanating from the technology development pipeline through to structure solution, analysis and deposition. In addition we propose to serve as a structural biology research resource for the greater biological community, forming a partnership with biologists and structural biologists that will make the high throughput crystallization and cryopreservation pipeline available as a community resource. We currently serve the needs of more than 300 structural biologists who provide proteins for high throughput screening of chemical conditions that foster crystallization. We will extend that pipeline to include optimization and growth of frozen, mounted, diffraction-quality crystals ready for structural analysis. Access to the pipeline will rapidly extend the benefits of the technology development to a larger biological community.
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