IN-SITU X-RAY CRYSTALLOGRAPHY FOR PROTEIN CRYSTALS GROWN IN MICROCAPILLARIES

微毛细管中生长的蛋白质晶体的原位 X 射线晶体学

• 批准号: 7726024
• 负责人: RUSTEM F ISMAGILOV
• 金额: $ 0.39万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7726024
• 关键词: Blood capillaries; Caliber; Computer Retrieval of Information on Scientific Projects Database; Condition; Consumption; Crystallization; Crystallography; Data Set; Diffusion; Funding; Glass; Grant; Hybrids; In Situ; Institution; KLK3 gene; Membrane Proteins; Methane; Methods; Microfluidics; Modeling; Oils; Particulate; Proteins; Publishing; Reaction; Research; Research Personnel; Resolution; Resources; Roentgen Rays; Sampling; Screening procedure; Source; Structure; System; Techniques; United States National Institutes of Health; X ray diffraction analysis; X-Ray Diffraction; base; beamline; capillary; mutant; nanolitre; novel; research study; vapor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Microfluidic approaches to macromolecular crystallization has resulted in greater efficiency in crystallization due to the reduced amount of sample consumption. In our method, crystallization trials are confined to plugs, which are surrounded by immiscible oil to carry out microbatch protein crystallization experiments. We have already shown that we can collect full data sets of crystals grown in microcapillaries (Bo Zheng, Joshua D. Tice, L. Spencer Roach, and Rustem F. Ismagilov, "A Droplet-Based, Composite PDMS/Glass Capillary Microfluidic System for Evaluating Protein Crystallization Conditions by Microbatch and Vapor-Diffusion Methods with On-Chip X-Ray Diffraction", Angew. Chem. Int. Edit. 2004 43: 2508-2511). We also developed a new hybrid method to simutaneously screen and optimize crystallization conditions for membrane proteins (Liang Li, Debarshi Mustafi, Qiang Fu, Valentina Tereshko, Delai L. Chen, Joshua D. Tice, Rustem F. Ismagilov, "Nanoliter microfluidic hybrid method for simultaneous screening and optimization validated with crystallization of membrane proteins", PNAS,2006,103: 19243-19248). The first complete data set was collected using in-situ x-ray diffraction on capillary grown crystals at GM/CA CAT beamline at the APS facility. The crystals were grown in 0.2 mm diameter Hampton Research capillaries at the Ismagilov lab with proteins provided by the Kuhn-Stevens labs at Scripps. We have been able to obtain X-ray defined structure of a novel soluble protein, oligoendopeptidase F, and a model membrane protein, reaction center and a new mutant sturcture of reaction center. All these crystals were grown using the microfluidic method. Moreover, now that our technique has been established, we are on the brink of solving many more crystal structures. In 2007-3, we were able to get full data sets at 3.6 A of a new membrane protein, particulate methane monooxydase from OB3B. We are pursuing higher resolution in order to publish it.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 由于样品消耗量减少，大分子结晶的微流体方法提高了结晶效率。在我们的方法中，结晶试验仅限于塞子，塞子被不混溶的油包围以进行微批量蛋白质结晶实验。我们已经证明，我们可以收集微毛细管中生长的晶体的完整数据集（Bo Cheng、Joshua D. Tice、L. Spencer Roach 和 Rustem F. Ismagilov，“基于液滴的复合 PDMS/玻璃毛细管微流体系统”）通过片上 X 射线衍射的微批量和蒸汽扩散方法评估蛋白质结晶条件”，Angew. Chem。国际编辑2004 43：2508-2511）。我们还开发了一种新的混合方法来同时筛选和优化膜蛋白的结晶条件（Liang Li，Debarshi Mustafi，Qiang Fu，Valentina Tereshko，Delai L. Chen，Joshua D. Tice，Rustem F. Ismagilov，“纳升微流体混合方法”用于通过膜蛋白结晶进行同步筛选和优化验证”，PNAS，2006，103： 19243-19248）。第一个完整的数据集是在 APS 设施的 GM/CA CAT 光束线上使用毛细管生长晶体的原位 X 射线衍射收集的。这些晶体在伊斯马吉洛夫实验室的汉普顿研究中心直径 0.2 毫米的毛细管中生长，蛋白质由斯克里普斯的库恩-史蒂文斯实验室提供。 我们已经能够获得一种新型可溶性蛋白质的 X 射线确定结构， 寡内肽酶F，以及模型膜蛋白、反应中心和反应中心的新突变结构。所有这些晶体都是使用微流体方法生长的。此外，既然我们的技术已经建立，我们即将解决更多的晶体结构。 2007 年 3 月，我们能够获得 OB3B 的新型膜蛋白颗粒甲烷单氧化酶 3.6 A 的完整数据集。我们正在追求更高的分辨率以便发布它。