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. We are determining the effectiveness of using high pressure freezing of crystals to provide cryo protection, without the need for conventional chemical cryo protection. This evaluation will be useful in deciding how we with to proceed with respect to transfering this technology to the PxRR and the NSLS for use by the general user community. We are working in collaboration with the group at CHESS that developed this technology, and with the NSLS. Crystals of cubic insulin and rhombohedral insulin were prepared and frozen under vaying conditions of pressure and cryo protectant concentration. This method has previously proven most useful in crystals with relatively low solvent content, and the very large difference in the solvent content of the two specimen types (30% for rhobohedral insulin and 70% for cubic insulin) should provide data on this trend. A secondary objective of this project is to evaluate the density of the amorphous ice in the solvent region. The 011 reflection of cubic insulin is highly sensitive to the density contrast in the specimen, and we will track its intensity as the pressure is raised.
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