CRYSTAL SCREENING

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。 本项目的目标是筛选和收集来自GST2和GST蛋白质晶体的高分辨率X射线衍射数据。GST2和GST是两个单独的项目，需要单独的结晶条件才能生产出高质量的晶体用于X射线数据收集。 GST2和GST蛋白属于一类新的谷胱甘肽转移酶，该酶仅在线虫门中发现，因此可能成为线虫防治的靶标。这类GST的成员不存在X射线结构，并且与其他GST类的低序列同源性降低了同源建模在合理开发抑制剂中的可能用处，这需要详细的活性部位知识。 用悬滴气相扩散法在MacCHESS上进行了结晶实验。结晶的努力导致了两种形式的晶体从GST2蛋白。晶体筛选是在康奈尔高能同步加速器(CHESS)A1站进行的，使用15%甘油作为低温保护剂，用于LN2冷流中的闪速冻结。其中一种类型的晶体产生的衍射图是不可能索引的，但第二种类型的晶体产生了高分辨率的衍射数据。X-射线衍射数据从单个冻结晶体中采集，分辨率下限为1.8A。这些数据是使用区域探测器系统公司的Quantum 210 CCD探测器和OxfordCryosSystems公司的冷冻冷却系统记录的。采用Denzo程序对数据进行处理和整理。 采用相同的悬滴气相扩散结晶法对GST蛋白进行结晶。国际象棋F1站筛选出极细小易碎的水晶。晶体浸泡在15%的MPD低温溶液中，在液氮冷流中闪冷。一个1.9A分辨率的完整数据集是从单晶体上收集的，使用的是来自Area Detector Systems的Quantum 4 CCD探测器和来自OxfordCryosSystems的冷冻冷却系统。GST蛋白的结构用Denzo程序进行数据简化和处理，用Amore和Refmac5程序(CCP4文档)进行分子置换和精细化。已检查并批准了蛋白质数据库沉积的坐标文件。 综上所述，来自GST2和GST项目的蛋白质晶体被筛选并用于在MacCHESS进行成功的X射线衍射实验，并收集了高分辨率的结晶学数据用于进一步的分析和结构阐明。

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 objective of this project was to screen and collect high-resolution X-ray diffraction data from GST2 and GST protein crystals. GST2 and GST are two single projects that required separate crystallization conditions in order to produce quality crystals for X-ray data collection. GST2 and GST protein belong to the new class of glutathione transferase which has only been found in the nematode phylum and therefore presents a possible target for nematode control. No X-ray structures exist for members of this GST class and low sequence identity with other GST classes diminishes the possible usefulness of homology modeling in the rational development of inhibitors, which requires detailed knowledge of the active site. Crystallization experiments were carried out at MacCHESS with the hanging-drop vapor-diffusion method. The crystallization efforts resulted in two forms of crystals from GST2 protein. Crystal screening, using 15% Glycerol as a cryo protectant for flash freezing in the LN2 cold stream, was performed at Cornell High-Energy Synchrotron Source (CHESS), A1 station. The diffraction pattern produced from one type of the crystals was impossible to index, but the second type of crystals resulted in high resolution diffraction data. X-ray diffraction data were collected to resolution limit of 1.8 A from a single frozen crystal. The data were recorded using the Quantum 210 CCD detector from Area Detector Systems and a cryocooling system from OxfordCryosystems. Denzo program was used to process and reduce the data. The same hanging-drop vapor-diffusion crystallization method was used in order to crystallize GST protein. Highly delicate, small and fragile crystals ware screened at CHESS, F1 station. Crystals were soaked in 15% MPD cryo solution and flash cooled in the LN2 cold stream. A complete data set at 1.9 A resolution was collected from a single crystal using a Quantum 4 CCD detector from Area Detector Systems and a cryocooling system from OxfordCryosystems. The structure of GST protein was solved using Denzo program for data reduction and processing, AMORE and Refmac5 programs (CCP4 Documentation) for molecular replacement and refinement. Coordinate file was checked and approved for Protein Data Bank deposition. In summary, protein crystals from GST2 and GST projects were screened and utilized to perform successful X-ray diffraction experiments at MacCHESS with high resolution crystallographic data collected for further analysis and structure elucidation.