High-Throughput, Capillary-Based Protein Crystallography

高通量、基于毛细管的蛋白质晶体学

• 批准号: 6944874
• 负责人: Deirdre R. Meldrum
• 金额: $ 15.07万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2006-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6944874
• 关键词: X ray crystallography; automated data processing; biotechnology; computer system design /evaluation; cryopreservation; crystallization; experimental designs; high throughput technology; method development; protein purification; proteins

DESCRIPTION (provided by applicant): Preparing well-diffracting crystals is the key step in biomacromolecular crystallography and is in particular a challenge for high-throughput structure determination projects of proteins as part of structural genomics and medicinal protein crystallography projects. Another challenge is to freeze the crystals obtained to 100K in such a manner that they remain well-diffracting and with no ice crystals. This is a critical step for X-ray data collection at synchrotron beam lines. The next critical step is to place the crystal in the X-ray beam and center the crystal precisely at the proper position where cryo-stream, X-rays and rotation axes of the goniometer intersect. The aim of our proposal is to obtain a completely automated procedure for all these steps: no manual intervention for crystal growth, cryofreezing and crystal centering. This unique approach can significantly remove all bottlenecks between protein production and the initiation of X-ray data collection for biomacromolecular crystallography. The equipment to be developed would be able to work with very small amounts of protein samples since volumes per experiment are in the low nanoliter range. The aim of the proposed project is to develop, design, and build a complete prototype sample processor that automates all process steps from initial protein sample, through automated detection of crystal growth, though delivery of a completely characterized, cryocooled sample to the synchrotron or other x-ray diffraction facility. Specific performance goals of this prototype system include: 1) A complete pipeline: sample preparation, sample sealing, conditioned storage for crystal growth, automated identification of crystal growth, cryocooling, and characterization of the samples in anticipation of x-ray crystallography. 2) Fully-automated, "hands-free" processing of samples. 3) Throughput of at least 500 samples/hour. 4) In-line image acquisition and processing for automated identification of crystal growth. The proposal has not only potential for a major impact of full automation of all steps between purified protein production and starting the X-ray data collection process in structural genomics projects. In addition, it has the potential to be coupled with combinatorial libraries of chemical compounds, which would allow thousands of compounds to be tested for crystal growth of a drug target protein in the presence of numerous different compounds.

描述（由申请人提供）：制备良好衍射的晶体是生物大分子晶体学的关键步骤，尤其是作为结构基因组学和药用蛋白质晶体学项目一部分的蛋白质高通量结构测定项目的挑战。另一个挑战是将获得的晶体冷冻至 100K，使其保持良好的衍射性且不含冰晶。这是同步加速器光束线 X 射线数据收集的关键步骤。下一个关键步骤是将晶体放置在 X 射线束中，并将晶体精确地居中于冷冻流、X 射线和测角仪旋转轴相交的正确位置。我们建议的目的是获得所有这些步骤的完全自动化程序：晶体生长、冷冻和晶体居中无需人工干预。这种独特的方法可以显着消除蛋白质生产和生物大分子晶体学 X 射线数据收集启动之间的所有瓶颈。待开发的设备将能够处理非常少量的蛋白质样品，因为每次实验的体积都在低纳升范围内。 拟议项目的目的是开发、设计和构建一个完整的原型样品处理器，该处理器可自动执行从初始蛋白质样品到晶体生长的自动检测，再到将完全表征的低温冷却样品传送到同步加速器或其他设备的所有处理步骤。 X射线衍射设备。该原型系统的具体性能目标包括： 1) 完整的流程：样品制备、样品密封、晶体生长的条件存储、晶体生长的自动识别、低温冷却以及 X 射线晶体学预测中的样品表征。 2) 全自动、“免提”样品处理。 3) 吞吐量至少为 500 个样本/小时。 4) 在线图像采集和处理，用于自动识别晶体生长。 该提案不仅有可能对结构基因组学项目中纯化蛋白质生产和启动 X 射线数据收集过程之间所有步骤的完全自动化产生重大影响。此外，它还有可能与化合物组合库相结合，这将允许在多种不同化合物存在的情况下测试数千种化合物的药物靶蛋白晶体生长情况。