Tools for High Throughput Structural Biology

描述（由申请人提供）：我们建议为高通量结构生物学组装技术开发和结构测定管道。我们的重点将放在衍射研究的样品制备上。我们的技术开发工作将集中在（1）从酵母中表达和纯化小蛋白-蛋白复合物;（2）从酵母中表达和纯化跨膜蛋白;（3）鉴定、表达和纯化靶向来自酵母的跨膜蛋白的抗体，作为其结晶的辅助;（4）蛋白质复合物、跨膜蛋白、跨膜蛋白-抗体复合物和可溶性蛋白的结晶;和（5）蛋白质晶体的冷冻保存。所有的技术开发工作都将致力于或最终致力于高吞吐量。我们的结构确定管道将以中等吞吐量运行，并将从技术开发管道开始进行项目，直到结构解决方案，分析和沉积。 此外，我们建议作为更大的生物界的结构生物学研究资源，与生物学家和结构生物学家建立伙伴关系，使高通量结晶和冷冻保存管道作为社区资源。我们目前服务于300多名结构生物学家的需求，他们为促进结晶的化学条件的高通量筛选提供蛋白质。我们将扩展该管道，包括优化和生长冷冻，安装，衍射质量晶体准备结构分析。进入管道将迅速扩大技术发展的好处，以更大的生物社区。

DESCRIPTION (provided by applicant): 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.