IMPROVING BROMIDE SOAKS FOR SOLVING THE PHASE PROBLEM IN CRYSTALLOGRAPHY

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。 有许多哺乳动物蛋白质不能用Se-蛋氨酸细菌表达系统生产。对于这些蛋白质，可以通过添加重原子来解决相问题，并利用它们的微分散射来获得相信息和产生电子密度。由Z.Dauter(NSLS)首创的卤化物浸渍方法是Se-Met相变的一种替代方法。用高浓度的溴或碘溶液短期浸泡蛋白质晶体会产生表面结合的离子。另一种通用的定相方法是通过加压蛋白质晶体(由SSRL开发)引入惰性气体，如氪和氙气。如果蛋白质有一些疏水空腔，惰性气体通常会结合在其中。遗憾的是，这些改进方法的效率较低。由于渗透压的不同，卤化物浸泡往往会对蛋白质晶格造成压力。惰性气体加压晶体通常会出现松散的衍射，并且比原始晶体具有更高的马赛克，这很可能是由于水合作用的变化。对溶菌酶(高分辨率；1.7A)、β-内酰胺酶(中分辨率；2.3A)和胸苷合成酶互补蛋白TM0449(低分辨率；3.8A)应用改进的浸泡方案以减少渗透或脱水的恶化影响。所有的晶体都用溴化锶(RbBr)和含甘油的溶液处理，得到了无冰的衍生冷冻晶体。除RbBr处理外，还用Kr加压一个溶菌酶晶体。这些元素可用于在SSRL波束线路9-1、9-2和11-1处进行MAD数据收集。对数据的初步分析表明：(A)在浸泡过程中保持了分辨率和嵌合性；(B)Rb和Br2+对所有蛋白质都有显著的结合；(C)高分辨率数据(溶菌酶)的自动完全建模，以及中分辨率数据(β-内酰胺酶)的部分建模。

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).