HIGH PRESSURE COOLING OF E COLI CLASS IA RIBONUCLEOTIDE REDUCTASE COMPLEX

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。 核糖核苷酸还原酶S是细胞内脱氧核糖核苷酸的唯一来源，对所有活细胞都是至关重要的。虽然核糖核苷酸还原酶有多种类型，但对大肠杆菌的I类酶已有了广泛的研究。该反应需要两种蛋白质，即含有α的活性部位和产生自由基的蛋白β。β为α的活性部位提供自由基，是催化所必需的。虽然这两个亚基的结构是已知的，但络合物中没有可用的α和β结构，活性络合物的齐聚状态也是未知的。在复合体中获得了α和β的晶体，以开始解决迄今为止使用的生物化学和生物物理方法尚未解决的一些问题。这些晶体的最佳衍射值约为5.5°。收集了一个5.8°的数据集，在这个分辨率下，用两个已知的亚基结构进行了分子置换，得到了结构。考虑到分辨率较低，这些地图的质量非常高。我们目前正在努力提高分辨率，以便获得关于我们在结构中确实看到的络合物的原子分辨率信息。在康奈尔大学的Sol M.Gruner博士和Chae Un Kim博士的合作下，我们正在尝试使用他们新开发的高压冷冻方法来提高我们晶体的分辨率。我们在CHESS请求光束时间，以测试在康奈尔大学进行的高压冷冻对这些晶体的衍射性质的影响，如果成功，将收集完整的本地数据集。

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. Ribonucleotide reductases (RNR¿¿"s) are the sole source of deoxyribonucletides in the cell an are thus vital for all living cells. While there are multiple classes of RNR¿¿"s the E. coli class I enzyme has been extensively studied. The reaction requires two proteins, the active site containing alpha and the radical generating protein beta. Beta provides a radical to the active site of alpha, required for catalysis. While structures of the two subunits are known, there is no available structure of alpha and beta in complex and the oligomerization state of the active complex is not known. Crystals were obtained of alpha and beta in complex to begin to address some of the issues unresolved by biochemical and biophysical methods used so far. The best diffraction seen for these crystals is around 5.5 ¿ . A 5.8 ¿ data set was collected and the structure was solved at this resolution by molecular replacement using the two know subunit structures. The maps are of very high quality considering the low resolution. We are currently trying to improve the resolution in order to obtain atomic resolution information about the complex we do see in the structure. In collaboration with Dr. Sol M. Gruner and Dr. Chae Un Kim at Cornell, we are attempting to improve the resolution of our crystals using their newly developed high-pressure cryocooling approach. We are requesting beam time at CHESS to test the effects of high-pressure cryocooling, carried out at Cornell, on the diffraction properties of these crystals, and if successful, collecting full native datasets.