ORT: A TEST CASE WITH THE ATP-DEPENDENT CHROMATIN REMODELING COMPLEX RSC

ORT：ATP 依赖性染色质重塑复合物 RSC 的测试案例

• 批准号: 7956451
• 负责人: Andres Leschziner
• 金额: $ 0.65万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7956451
• 关键词: ATP Hydrolysis; Address; Automation; Behavior; Chromatin; Chromatin Remodeling Factor; Complex; Complication; Computer Retrieval of Information on Scientific Projects Database; Cryoelectron Microscopy; DNA; Data; Data Collection; Funding; Goals; Grant; Histones; Image; Institution; Manuals; Methods; Microscopy; Modeling; Molecular; Negative Staining; Nucleosomes; Pathway interactions; Research; Research Personnel; Resolution; Resources; Sampling; Source; Staining method; Stains; Testing; United States National Institutes of Health; Yeasts; chromatin remodeling; flexibility; interest; macromolecule; particle; reconstruction

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. A variety of cellular factors are involved in regulating the dynamic behavior of chromatin, thus making specific DNA segments available, or unavailable, according to specific needs. Among them are the ATP-dependent chromatin remodeling factors, large, multisubunit complexes that use the energy from ATP hydrolysis to non-covalently modify histone-DNA contacts. Despite a large body of experimental data we do not yet understand the mechanism by which these macromolecules perform their function. We are taking a structural approach to address this problem with the goal of eventually visualizing a number of intermediates along the remodeling pathway at high resolution using cryo-electron microscopy (cryo-EM) and single-particle methods. In this particular instance, we are interested in testing the recently developed automation of data collection using the Orthogonal Tilt Reconstruction (OTR) method (which we developed) as has been incorporated into Leginon. Obtaining correct initial models for single particles by (cryo-)EM remains one of the major challenges in the field, particularly for asymmetric particles that are conformationally flexible (as it appears to be the case for these large chromatin remodelers--see (d) below). The ability to bring automated data collection to this problem would be invaluable. Furthermore, successful automated data collection of images from tilted samples under cryo conditions would allow for the application of the OTR method directly to vitrified samples, thus removing the additional complication of flattening associated with the negatively stained samples often used to obtain initial reconstructions. We have previously obtained initial reconstructions of the yeast S.cerevisiae chromatin remodeling RSC in negative stain using the OTR method and manual data collection. The goal for this project is to obtain initial reconstructions of a RSC:nucleosome complex in negative stain (and possibly vitrified) as well as an initial reconstruction of RSC under vitrified conditions in order to compare it with the one we previously obtained from a negatively stained sample.

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。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 多种细胞因素参与调节染色质的动态行为，从而根据特定的需要使特定的 DNA 片段可用或不可用。其中包括 ATP 依赖性染色质重塑因子，这是一种大型多亚基复合物，利用 ATP 水解产生的能量来非共价修饰组蛋白-DNA 接触。尽管有大量的实验数据，我们仍然不了解这些大分子发挥其功能的机制。我们正在采取结构性方法来解决这个问题，目标是最终使用冷冻电子显微镜（cryo-EM）和单粒子方法以高分辨率可视化重塑途径中的许多中间体。 在这个特殊的例子中，我们有兴趣使用正交倾斜重建（OTR）方法（我们开发的）来测试最近开发的数据收集自动化，该方法已被纳入 Leginon 中。通过（冷冻）EM 获得单个颗粒的正确初始模型仍然是该领域的主要挑战之一，特别是对于构象灵活的不对称颗粒（这些大型染色质重塑剂似乎就是这种情况 - 参见下面的 (d)）。自动化数据收集解决这个问题的能力将是无价的。此外，在低温条件下成功地自动收集倾斜样品的图像数据将允许将 OTR 方法直接应用于玻璃化样品，从而消除与经常用于获得初始重建的负染色样品相关的扁平化的额外复杂性。我们之前已经使用 OTR 方法和手动数据收集在负染色中获得了酿酒酵母染色质重塑 RSC 的初步重建。该项目的目标是获得负染色（可能是玻璃化）中 RSC：核小体复合物的初始重建，以及玻璃化条件下 RSC 的初始重建，以便将其与我们之前从负染色样品中获得的复合物进行比较。