CRYSTAL SCREENING

点击翻译按钮获取中文摘要

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.