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。 核糖核苷酸还原酶（RNR）是细胞中脱氧核糖核苷酸的唯一来源，因此对所有活细胞都至关重要。虽然有多个类的RNR的E。大肠杆菌I类酶已被广泛研究。该反应需要两种蛋白质，含有α的活性位点和产生蛋白质β的自由基。β为α的活性位点提供了一个自由基，这是催化所必需的。虽然这两个亚基的结构是已知的，但复合物中没有α和β的可用结构，并且活性复合物的寡聚化状态也是未知的。晶体获得的α和β在复杂的开始，以解决一些问题尚未解决的生化和生物物理方法使用至今。 这些晶体的最佳衍射约为5.5 º。收集了5.8 ½的数据集，并使用两种已知的亚基结构通过分子置换在该分辨率下解析了结构。考虑到低分辨率，这些地图的质量非常高。我们目前正试图提高分辨率，以获得我们在结构中看到的复合物的原子分辨率信息。 与Sol M博士合作。Gruner和康奈尔大学的Chae Un Kim博士，我们正试图使用他们新开发的高压低温冷却方法来提高晶体的分辨率。我们正在请求CHESS的光束时间来测试康奈尔大学进行的高压低温冷却对这些晶体衍射特性的影响，如果成功，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.