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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 微流控大分子结晶的方法由于减少了样品消耗量而导致了更高的结晶效率。在我们的方法中，结晶实验被限制在塞子上，塞子被不混溶的油包围，以进行微批蛋白质结晶实验。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 Zeng，Joshua D.Tice，L.Spencer Roach，和Rustef.Ismagilov，“一种基于液滴的，复合PDMS/玻璃毛细管微流体系统，用于通过微批处理和气相扩散方法与芯片上X射线衍射评估蛋白质结晶条件”，Angew。化学。内部编辑。2004年43：2508-2511)。我们还开发了一种新的杂交法来同时筛选和优化膜蛋白的结晶条件(梁丽，Debarshi Mustafi，Qiang Fu，Watina Tereshko，Delai L.Chen，Joshua D.Tice，Rustef.Ismagilov，“纳升微流控杂交法用于同时筛选和优化膜蛋白的结晶”，PNAS，2006,103：19243-19248)。第一个完整的数据集是在APS设施的GM/CA CAT光束线上对毛细生长的晶体进行原位X射线衍射收集的。这些晶体是在伊斯马吉洛夫实验室的0.2毫米直径的汉普顿研究毛细管中生长的，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的可溶性蛋白质的X射线定义结构， 寡肽内切酶F和一个模型膜蛋白、反应中心和一个新的突变反应中心结构。所有这些晶体都是用微流体法生长的。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。在2007-2003年，我们能够从OB3B获得一种新的膜蛋白，颗粒甲烷单加氧酶的完整数据集，温度为3.6A。我们正在追求更高的分辨率，以便出版它。

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.