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: 7725992
• 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/7725992
• 关键词: Blood capillaries; Caliber; Complex; 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; Methods; Microfluidics; Modeling; Oils; Proteins; Reaction; Research; Research Personnel; 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. We have crystallized three novel membrane proteins in our system and some very important complexes of soluble proteins. The upcoming beamtime would allow us to move ahead and solve the new structures.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 大分子结晶的微流体方法由于样品消耗量的减少而导致更高的结晶效率。在我们的方法中，结晶试验仅限于塞，这是由不混溶的油包围进行微批量蛋白质结晶实验。我们已经证明，我们可以收集在微毛细管中生长的晶体的完整数据集（Bo Zheng，约书亚D.泰斯湖Spencer Roach和Rustem F. Ismagilov，“一种基于液滴的复合PDMS/玻璃毛细管微流控系统，用于通过微批处理和气相扩散方法与片上X射线衍射评估蛋白质结晶条件”，Angew.化学国际编辑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，约书亚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）。第一个完整的数据集收集使用原位X射线衍射毛细管生长的晶体在GM/CA CAT光束线在APS设施。晶体在Ismagilov实验室的直径为0.2 mm的汉普顿研究毛细管中生长，蛋白质由Scripps的Kuhn-Stevens实验室提供。 我们已经能够获得一种新的可溶性蛋白质的X射线确定的结构， 寡肽内肽酶F和一种模式膜蛋白、反应中心和一种新的突变型反应中心结构。所有这些晶体都是使用微流体方法生长的。此外，现在我们的技术已经建立，我们即将解决更多的晶体结构。我们在我们的系统中结晶了三种新的膜蛋白和一些非常重要的可溶性蛋白复合物。即将到来的光束时间将使我们能够向前迈进，解决新的结构。