SOLUTION SCATTERING STUDIES ON PROTEIN COMPLEXES IN YEAST TRANSCRIPTION

酵母转录中蛋白质复合物的溶液散射研究

• 批准号: 7598030
• 负责人: Yuichiro Takagi
• 金额: $ 0.16万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7598030
• 关键词: Binding; Complex; Computer Retrieval of Information on Scientific Projects Database; Condition; DNA; Data; Data Set; Dimensions; Electron Microscopy; Elements; Funding; Genetic Transcription; Grant; Head; Human; Institution; Length; Mediator of activation protein; Modeling; Organism; Phase; Polymerase; Proteins; RNA Polymerase II; Radiation; Range; Research; Research Personnel; Resolution; Resources; Sampling; Shapes; Solutions; Source; Structure; Testing; Time; United States National Institutes of Health; Yeasts; dalton; molecular mass; particle; radius bone structure; three-dimensional modeling

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. Mediator is a central regulatory complex in transcription, and transmits information from activators and repressors bound at the regulatory DNA elements to RNA polymerase II (Pol II) in the entire range of organisms from yeast to humans. Mediator is composed of a total of 21 subunits with a molecular mass of over a million Dalton. We have successfully co-expressed all seven subunits of the head module of Mediator (223 kDa), and purified it in a large quantity. We have recently obtained crystals diffracting up to 4 ¿, and a full native data set at 5.6 ¿. We turned to small angle x-ray scattering to obtain an initial phase model for the crystal structure determination as well as to study the full-length Mediator, its subcomponents such as the head module, as well as the Mediator-Pol II complex. We have recently recorded solution x-ray scattering data to test feasibility of obtaining a structure at intermediary resolution. The Mediator head module sample in an optimal condition suffered little radiation damage. We obtained the radius of gyration 52 ¿, the maximum particle dimension of 165 ¿. We have been able to obtain a three dimensional model using Dammin and data recorded up to Q~0.26 ¿-1. Remarkably, the shape of the solution structure is consistent with the structure of the head module of Mediator determined by electron microscopy and single particle analysis. More recently, we obtained beam time on our rapid access proposal, and extended the structural resolution to approximately 10 ¿. One of the structural envelopes obtained by Gasbor using the most recent data has given an encouraging result in the crystallographic phase extension using Phaser.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 对于中心来说，它不一定是研究者的机构。 介体是转录中的中心调节复合体，在从酵母到人类的整个生物体中，将来自与调节 DNA 元件结合的激活子和阻遏子的信息传递给 RNA 聚合酶 II (Pol II)。介体总共由 21 个亚基组成。我们已经成功地共表达了Mediator头部模块的全部七个亚基（223 kDa），并且最近获得了大量的晶体。衍射高达 4 ¿ ，以及 5.6 ¿ 的完整本机数据集我们利用小角度 X 射线散射来获得用于确定晶体结构的初始相模型，并研究全长 Mediator、其子组件（例如头模块）以及 Mediator-Pol II 复合物。最近记录了溶液 X 射线散射数据，以测试在最佳条件下获得中等分辨率结构的可行性，我们获得了旋转半径 52 ¿ ，最大颗粒尺寸 165 ¿我们已经能够使用 Dammin 获得三维模型并记录高达 Q~0.26 ¿值得注意的是，解决方案结构的形状与通过电子显微镜和单粒子分析确定的 Mediator 头部模块的结构一致，最近，我们在快速访问提案中获得了束流时间，并将结构分辨率扩展到了。大约 10 ¿ Gasbor 使用最新数据获得的结构包络之一在使用 Phaser 的晶体相扩展中给出了令人鼓舞的结果。