Enabling membrane protein structural analysis: Tools for capillary diffusion crystallization and remote in situ diffraction experiments

项目摘要 这项提议是开发一种基于毛细管反扩散的综合方法来进行原位采集 大分子的衍射数据。将要开发的方法将使低温和常温 温度数据收集，后者对基于结构的药物设计至关重要。方法是 基于预装在支架中的毛细管，该支架便于将其从装载移至晶体 从生长到衍射分析，无需物理接触或直接操纵晶体。这个 毛细管一端封闭，通过离心法填充，这种方法可以使填充量非常高。 粘度溶液。结晶是通过毛细管反扩散(CCD)进行的，这种方法通常会导致 填充毛细管内径的晶体的生长，通常是多个晶体。 这项工作的直接研究目标是开发和改进坐骑 毛细管设计。这些更具体地集中在毛细管支架的设计上，即毛细管是如何匹配的 对于持有者来说，可以使用的毛细内径的尺寸下限，毛细材料 这是可以使用的，以及将毛细管固定到支架上的方法。不同的溶液器 将安装的毛细管引入沉淀剂溶液进行结晶的设计 被调查的，以及用于离心填充毛细管的夹持器。随着设计的实施 然后，通过可溶性和完整膜蛋白的生长和随后的衍射来测试它们 水晶。 这项提议是国际和平研究所、水晶定位系统公司的理查德·豪厄尔斯先生、 阿拉巴马大学亨茨维尔分校的马克·普西博士和斯坦福大学同步辐射的艾娜·科恩博士 实验室。RH将负责所有机械车间的工作，按照MP建议的设计制造零件 和交流。MP将负责组装安装的毛细血管，准备要使用的蛋白质 作为测试材料，搭建了结晶实验，并对所研制的硬件进行了各项功能测试。 AC将负责这项工作的所有光束线特定方面；测试安装在 并确保它们与同步加速器源中使用的样品交换机器人兼容。 衍射数据收集和数据分析将使用SSRL光束线和计算基础设施。数据和 结构分析将由RH监督，并由MP和AC提供输入。AC和AH将与MP一起提供 向RH输入以改进设计，然后在制造时对这些改进进行测试。

Project Summary This proposal is to develop an integrated capillary counter diffusion-based approach to in situ collection of macromolecule diffraction data. The method to be developed will enable both cryogenic and ambient temperature data collection, with the latter being critical to structure-based drug design. The approach is based on a capillary that is pre-mounted in a holder that facilitates its being moved from loading to crystal growth to diffraction analysis without ever having to physically touch or directly manipulate the crystal. The capillary is closed at one end and is filled by centrifugation, which method enables filling with very high viscosity solutions. Crystallization is by capillary counter diffusion (CCD), a method that often results in the growth of crystals, often multiple crystals, that fill the internal diameter of the capillary. The immediate research goals of this effort are the development of and improvements to the mounted capillary design. These are more specifically focused on the capillary holder design, how the capillary is mated to the holder, the lower size limits for the capillary internal diameter that can be employed, the capillary materials that can be used, and the methodology by which the capillary is secured to the holder. Different solution holder designs, by which the mounted capillary is introduced to the precipitant solution for crystallization, will be investigated, as well as the holder used for centrifugally filling the capillary. As the designs are implemented they will then be tested by the growth and subsequent diffraction of soluble and integral membrane protein crystals. This proposal is a collaborative effort between the PI, Mr. Richard Howells of Crystal Positioning Systems, Dr. Marc Pusey of the University of Alabama in Huntsville, and Dr. Aina Cohen of Stanford Synchrotron Radiation Laboratory. RH will be responsible for all machine shop work, fabricating parts to designs as suggested by MP and AC. MP will be responsible for assembly of the mounted capillaries, preparation of the proteins to be used as test materials, setting up the crystallization experiments, and all functional testing of the hardware developed. AC will be responsible for all beamline-specific aspects of this effort; to test the mounted capillaries on the beamline and to ensure their compatibility with sample exchange robots employed at synchrotron sources. Diffraction data collection and data analysis will use the SSRL beamlines and computing infrastructure. Data and structure analysis will be overseen by RH with input from MP and AC. AC and AH, together with MP, will provide input to RH for improvements to the designs, and will then test those improvements as they are fabricated.