IMPROVING BROMIDE SOAKS FOR SOLVING THE PHASE PROBLEM IN CRYSTALLOGRAPHY

改进溴化物浸泡解决晶体学中的相问题

• 批准号: 7954294
• 负责人: Tzanko Doukov
• 金额: $ 1.04万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7954294
• 关键词: Binding; Bromides; Bromine; Computer Retrieval of Information on Scientific Projects Database; Crystallography; Data; Data Analyses; Data Collection; Dehydration; Elements; Freezing; Funding; Glycerol; Grant; Heat shock proteins; Hydration status; Ice; Institution; Iodine; Ions; Krypton; Methionine; Methods; Modeling; Modification; Muramidase; Noble Gases; Osmosis; Osmotic Pressure; Phase; Procedures; Proteins; Research; Research Personnel; Resolution; Resources; Rubidium; Scheme; Solutions; Source; Surface; System; Thymidylate Synthase; United States National Institutes of Health; Xenon; beta-Lactamase; electron density; improved; meetings; stress protein; structural biology; synchrotron radiation

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. There are number of mammalian proteins that cannot be produced with Se-Methionine bacterial expression systems. For those proteins the phase problem could be solved by adding heavy atoms and use their differential scattering to obtain phase information and generate electron density. The method of halide soaking pioneered by Z. Dauter (NSLS) is one alternative to the Se-Met phasing. Short soaking of the protein crystals with high concentration bromine or iodine solutions results in a surface bound ions. Another universal phasing method is to introduce inert gases such as krypton and xenon by pressurizing protein crystals (developed at SSRL). If the proteins have some hydrophobic cavities the inert gases typically bind in them. Unfortunately, these modification methods suffer from low efficiency. Halide soaking often stresses the protein crystal lattice due to differences in osmotic pressure. Inert gas pressurized crystals often loose diffraction and have higher mosaicity than the initial crystals, mostly likely due to changes in hydration. An improved soaking scheme guided toward decreasing the deteriorating effects of osmosis or dehydration was applied to lysozyme (high resolution; 1.7 A), beta lactamase (medium resolution; 2.3 A), and thymidylate synthase complementary protein TM0449 (low resolution; 3.8 A). All crystals were treated with a rubidium bromide (RbBr) and glycerol containing solution resulting in ice-free derivatized frozen crystals. One lysozyme crystal was pressurized with Kr in addition to the RbBr treatment. These elements are accessible for MAD data collection at SSRL beam lines 9-1, 9-2, and 11-1. Preliminary analysis of the data indicates there is: (a) preserved resolution and mosaicity during the soak procedure; (b) significant binding of Rb and Br ions for all proteins; (c) automatic full model building for the high resolution data (lysozyme), and partial model building for the medium resolution data (beta lactamase).

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。 有许多哺乳动物蛋白不能用硒-蛋氨酸细菌表达系统产生。对于这些蛋白质，相位问题可以通过添加重原子并利用它们的微分散射来获得相位信息并产生电子密度来解决。本文介绍了Z. Dauter（NSLS）是Se-Met定相的一种替代方法。用高浓度溴或碘溶液短时间浸泡蛋白质晶体会产生表面结合离子。另一种通用的定相方法是通过加压蛋白质晶体引入惰性气体，如氪和氙（由SSRL开发）。如果蛋白质有一些疏水空腔，惰性气体通常会结合在其中。不幸的是，这些改性方法效率低。由于渗透压的差异，卤化物浸泡通常会对蛋白质晶格产生应力。惰性气体加压的晶体通常松散衍射，并且具有比初始晶体更高的镶嵌性，这很可能是由于水合作用的变化。 对溶菌酶（高分辨率; 1.7 A）、β-内酰胺酶（中等分辨率; 2.3 A）和胸苷酸合成酶互补蛋白TM 0449（低分辨率; 3.8 A）应用了针对降低渗透或脱水的劣化效应的改进的浸泡方案。所有晶体用含有溴化铷（RbBr）和甘油的溶液处理，得到无冰衍生的冷冻晶体。一个溶菌酶晶体加压与氪除了溴化铷处理。这些元件可用于在SSRL射束线9-1、9-2和11-1处的MAD数据收集。数据的初步分析表明：（a）在浸泡过程中保留了分辨率和镶嵌性;（B）所有蛋白质的Rb和Br离子的显著结合;（c）高分辨率数据（溶菌酶）的自动全模型构建和中分辨率数据（β-内酰胺酶）的部分模型构建。