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

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。 多种细胞因子参与调节染色质的动态行为，从而根据特定需要使特定的DNA片段可用或不可用。其中包括依赖于三磷酸腺苷的染色质重塑因子，这是一种大的、多亚单位的复合体，它利用三磷酸腺苷水解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，但我们仍不了解这些大分子发挥作用的机制。我们正在采取结构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显示重塑路径上的一些中间体。 在这种特殊情况下，我们感兴趣的是测试最近开发的使用(我们开发的)正交倾斜重建(OTR)方法的数据收集自动化，因为该方法已并入Leginon。通过(低温)EM获得单个粒子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粒子(就像这些大型染色质重构体的情况--见下文(D))。为这个问题带来自动化数据收集的能力将是无价的。此外，在低温条件下从倾斜的样品中成功地自动收集图像将允许将OTR方法直接应用于玻璃化样品，从而消除了与通常用于获得初始重建的负染样品相关的扁平化的额外复杂性。我们以前已经用OTR方法和人工数据收集获得了阴性染色的酵母染色质重塑RSC的初步重建。该项目的目标是获得RSC的初步重建：负染色核小体复合体(可能是玻璃化的)，以及在玻璃化条件下的RSC的初步重建，以便与我们以前从负染样本中获得的RSC进行比较。

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.