HIGH PRESSURE CRYOCOOLING AND RADIATION DAMAGE

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 生物样品的辐射损伤是目前高分子X射线晶体学的主要限制因素之一。在室温下减少辐射损伤的最著名方法之一是将晶体快速冷冻至低温，这通常需要寻找渗透性冷冻保护剂。最近开发了一种新的晶体冷冻方法，即高压冷冻冷却，通过在高压（1000 ~ 2000 atm）下冷冻晶体来避免使用冷冻保护剂。该机制被认为涉及高密度无定形（HDA）冰，其在环境压力下的密度为 1.17 g/cm3。 由于二次辐射损伤很大程度上取决于 X 射线辐射产生的自由基的扩散，因此与通常在环境压力下在晶体内部形成的低密度无定形冰相比，HDA 冰具有高粘度和密度，因此可能有效减少辐射损伤。 我们将测试高压低温冷却对辐射损伤的影响。在这项研究中，将在几种不同的压力（1 atm ~ 4000 atm）和不同的化学条件下制备高质量的蛋白质晶体。

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. Radiation damage to biological samples is currently one of the major limiting factors in macromolecular x-ray crystallography. One of the best known method to reduce radiation damage at room temperature is flash freezing crystals to cryogenic temperature, which often requires finding penetrating cryoprotectants. Recently a new crystal freezing method, high pressure cryocooling, was developed, where using of cryoprotectants was avoided by freezing crystals under high pressures (1000 ~ 2000 atm). The mechanism was suggested involving high density amorphous (HDA) ice which has density of 1.17 g/cm3 at ambient pressure. Since the secondary radiation damage highly depends on the diffusion of the free radicals produced by x-ray radiation, HDA ice might be effective to reduce radiation damage due to its high viscosity and density compared to low density amorphous ice that usually forms inside crystal at ambient pressure. We will test the effect of high pressure cryocooling on radiation damage. For the study, high quality protein crystals will be prepared at several different pressures(1 atm ~ 4000 atm) and different chemical conditions.