HIGH PRESSURE CRYOCOOLING IN PREPARATION FOR 70S RIBOSOME CRYSTALS

高压低温冷却制备 70S 核糖体晶体

• 批准号: 7721303
• 负责人: SOL M GRUNER
• 金额: $ 3.33万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7721303
• 关键词: Affect; Anodes; Biological Models; Cells; Chemicals; Computer Retrieval of Information on Scientific Projects Database; Data Collection; Elastases; Freezing; Funding; Grant; Ice; Image; Institution; Modeling; Pancreatic Elastase; Preparation; Process; Proteins; Protocols documentation; Purpose; Research; Research Personnel; Resources; Ribosomes; Solutions; Source; Structure; Temperature; United States National Institutes of Health; density; pressure; research study

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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 这些实验的主要目的是研究在制备低温冷却70 S核糖体晶体中的高压低温冷却过程的方面。这涉及通过应用于模拟蛋白质晶体来形成晶体中的高密度无定形（HDA）冰（Gruner组任务）的基础研究，以及由Noller组提供的关于压力冷冻70 S核糖体晶体的数据收集。 高压低温冷却将应用于三个不同的方向。在所有情况下，这些实验不能在旋转阳极源处进行。 1.涉及HDA冰的机制研究。 提出了高密度非晶（HDA）冰的高压低温冷却机制，HDA冰在1bar和77 K下的密度为1.17g/cm ~ 3。我们想证明HDA冰确实是通过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收集溶液/晶体衍射图像。 2.压力引起的结构变化通常很小，但有时不可忽略。我们最近观察到，弹性蛋白酶在压力冷冻时显示出显著的晶体晶胞变化（a= 50 A -> a= 46.5 A），并且其结构与常压下的结构有很大不同。更有趣的是，改变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 S核糖体晶体将使用各种方案进行压力冷冻并进行检查。