HIGH PRESSURE EXPERIMENTS WITH CUBIC INSULIN AND RHOMBOHEDRAL INSULIN

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。我们正在确定使用高压冷冻晶体来提供低温保护的有效性，而不需要常规的化学低温保护。这项评估将有助于决定我们如何将这项技术转移到PxRR和NSLS供一般用户社区使用。我们正在与开发这项技术的国际象棋小组以及NSLS合作。制备了立方胰岛素和菱面体胰岛素晶体，并在不同的压力和冷冻保护剂浓度条件下冷冻。该方法以前已被证明在溶剂含量相对较低的晶体中最有用，并且两种样品类型的溶剂含量差异非常大（菱面体胰岛素为30%，立方胰岛素为70%），应提供关于该趋势的数据。 该项目的第二个目标是评估溶剂区无定形冰的密度。立方胰岛素的011反射对样品中的密度对比度高度敏感，我们将随着压力的升高跟踪其强度。

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.