High-Throughput, Capillary-Based Protein Crystallography

描述(申请人提供)：制备衍射性良好的晶体是生物大分子结晶学的关键步骤，尤其是对作为结构基因组学和药物蛋白质结晶学项目一部分的高通量蛋白质结构确定项目来说是一个挑战。另一个挑战是将获得的晶体冻结到100K，使它们保持良好的衍射性，并且没有冰晶。这是在同步加速器束线上收集X射线数据的关键步骤。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在低温气流、X射线和测角仪旋转轴相交的适当位置。我们建议的目的是为所有这些步骤获得一个完全自动化的程序：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著消除蛋白质生产和生物大分子结晶学X射线数据收集之间的所有瓶颈。由于每个实验的体积都在低纳升范围内，因此将要开发的设备将能够处理非常少量的蛋白质样本。拟议项目的目标是开发、设计和构建一个完整的原型样品处理器，通过将完全表征的低温冷却样品交付到同步加速器或其他X射线衍射设备，实现从初始蛋白质样品到晶体生长的自动检测的所有过程步骤的自动化。该原型系统的具体性能目标包括：1)完整的流水线：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、低温冷却和样品的表征，以期进行X射线结晶学研究。2)全自动、“免提”的样品处理。3)至少500个样本/小时的吞吐量。4)用于自动识别晶体生长的在线图像采集和处理。该提议不仅有可能对结构基因组学项目中从纯化蛋白生产到启动X射线数据收集过程的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的组合库相结合，这将允许在存在大量不同化合物的情况下测试数千种化合物的药物靶蛋白的晶体生长。

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.