CRYSTAL SCREENING

晶体筛选

• 批准号: 8171487
• 负责人: DOLETHA SZEBENYI
• 金额: $ 27.63万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171487
• 关键词: Active Sites; Area; Computer Retrieval of Information on Scientific Projects Database; Crystallization; Data; Data Collection; Data Set; Databases; Deposition; Development; Diffusion; Documentation; Drops; Freezing; Funding; Glutathione S-Transferase; Glycerol; Grant; Homology Modeling; Institution; Knowledge; Methods; Molecular; Nematoda; Pattern; Process; Proteins; Research; Research Personnel; Resolution; Resources; Roentgen Rays; Screening procedure; Solutions; Source; Stream; Structure; Synchrotrons; System; United States National Institutes of Health; X ray diffraction analysis; X-Ray Diffraction; detector; indexing; inhibitor/antagonist; member; programs; quantum; research study; vapor

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.

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。 该项目的目的是筛选和收集GST 2和GST蛋白晶体的高分辨率X射线衍射数据。GST 2和GST是两个单独的项目，需要单独的结晶条件，以生产用于X射线数据收集的优质晶体。 GST 2和GST蛋白属于仅在线虫门中发现的新型谷胱甘肽转移酶，因此呈现了线虫控制的可能靶标。没有X-射线结构存在的成员，这GST类和低序列同一性与其他GST类减少了可能的有用性的同源性建模的抑制剂，这需要详细的知识的活性位点的合理发展。 在MacCHESS用悬滴气相扩散法进行结晶实验。结晶的努力导致两种形式的晶体从GST 2蛋白。在康奈尔高能同步加速器源（CHESS）A1站进行晶体筛选，使用15%甘油作为低温保护剂，用于在液氮冷流中快速冷冻。 从一种类型的晶体产生的衍射图案是不可能索引的，但第二种类型的晶体产生了高分辨率的衍射数据。从单个冷冻晶体收集X射线衍射数据至1.8 A的分辨率极限。使用来自Area Detector Systems的Quantum 210 CCD检测器和来自OxfordCryosystems的低温冷却系统记录数据。采用Denzo程序对数据进行处理和归约。 使用相同的悬滴气相扩散结晶方法来结晶GST蛋白。在国际象棋F1站筛选高度精致、小而易碎的晶体。 将晶体浸泡在15%MPD低温溶液中，并在液氮冷流中快速冷却。使用来自Area Detector Systems的Quantum 4 CCD检测器和来自OxfordCryosystems的低温冷却系统从单晶收集1.9 A分辨率的完整数据集。GST蛋白的结构用Denzo程序进行数据简化和处理，AMORE和Refmac 5程序（CCP 4 Documentation）进行分子置换和精修。检查并批准坐标文件用于蛋白质数据库沉积。 总之，GST 2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，收集高分辨率晶体学数据用于进一步分析和结构阐明。