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.

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