HIGH PRESSURE CRYOCOOLING IN PREPARATION FOR 70S RIBOSOME CRYSTALS

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。 这些实验的主要目的是研究高压冷冻过程的各个方面，为低温冷却70S核糖体晶体做准备。这包括通过应用于蛋白质晶体建模来在晶体中形成高密度无定形(HDA)冰的基础研究(Gruner小组任务)，以及由Noller小组提供的关于压力冻结的70秒核糖体晶体的数据收集。 高压冷冻将在三个不同的方向上应用。在所有情况下，这些实验都不能在旋转阳极源上进行。 1.HDA冰的作用机理研究。提出了高密度无定形(HDA)冰的高压低温冷却机制，高密度无定形冰在1bar和77K时的密度为1.17g/cm~3。我们想要证明HDA冰确实是由高压低温冷却形成的，并影响晶体衍射。我们计划通过逐渐升高温度来采集溶液/晶体的衍射图。 2.压力引起的结构变化通常很小，但有时不能忽略。我们最近观察到，当压力冻结时，弹性酶显示出明显的晶胞变化(a=50A--&gt；a=46.5A)，其结构与常压有很大的不同。更有趣的是，改变后的结构与化学微扰引起的结构非常相似。我们计划研究弹性蛋白酶作为一个模型系统，以了解压力对蛋白质的影响。 3.70年代的核糖体晶体将使用各种方案进行加压冷冻并进行检查。

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. The primary purpose of these experiments is to study aspects of the high pressure cryocooling process in preparation for cryocooling 70S ribosome crystals. This involves basic studies of the formation of high density amorphous(HDA) ice in crystals (Gruner group task) by application to model protein crystals, and data collection on pressure frozen 70S ribosome crystals provided by the Noller group. High pressure cryocooling will be applied in three different directions. In all cases, these experiments cannot be performed at rotating anode sources. 1. Mechanism study involving HDA ice. The mechanism of high pressure cryocooling was proposed involving high density amorphous (HDA) ice, which has 1.17 g/cm3 at 1 bar and 77 K. We would like to prove that HDA ice does form by high pressure cryocooling and affect crystal diffraction. We plan to collect solution/crystal diffraction images by increasing temperature gradually. 2. The structural changes induced by pressure are usually very small but sometimes not negligible. We recently observed that elastase showed a significant crystal unit cell change (a= 50 A --> a= 46.5 A) when pressure frozen and the structure was quite different than the ambient pressure one. More interestingly, the changed one is very similar to the structure induced by chemical perturbation. We plan to study elastase as a model system to understand pressure effects on proteins. 3. 70S ribosome crystals will be pressure frozen using various protocols and examined.