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

该子项目是利用 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来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 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DNA片段可用或不可用。其中包括ATP依赖性染色质重塑因子，即利用ATP水解产生的能量非共价修饰组蛋白-DNA接触的大型多亚基复合物。尽管有大量的实验数据，我们还不了解这些大分子发挥其功能的机制。我们正在采取结构的方法来解决这个问题，最终可视化的中间体沿着重塑途径在高分辨率使用冷冻电子显微镜（cryo-EM）和单粒子方法的目标。 在这个特定的例子中，我们有兴趣测试最近开发的自动化数据收集使用正交重构（OTR）方法（我们开发的）已被纳入Leginon。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，特别是对于构象柔性的不对称颗粒（因为对于这些大的染色质重塑物似乎是这种情况-参见下面的（d））。将自动化数据收集应用于这一问题的能力将是无价的。此外，在低温条件下从倾斜样品成功地自动收集图像数据将允许将OTR方法直接应用于玻璃化样品，从而消除与通常用于获得初始重建的负染色样品相关的平坦化的额外复杂性。我们以前已经获得了初步重建的酵母酿酒酵母染色质重塑RSC在负染色使用OTR方法和手动数据收集。该项目的目标是获得负染色（可能是玻璃化）的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，以便将其与我们先前从负染色样本中获得的重建进行比较。

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.