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

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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). 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. This unique technique has already become instrumental in crystallizing scarce targets and those that are difficult to crystallize. In addition to soluble proteins and RNA constructs, we have also been able to crystallize membrane proteins using our microfluidic set-up. We propose to use the synchotron at APS to obtain X-ray structures of these targets. The synochotron is necessary for small crystals that grow in microcapillaries.