Experimentally Guided Ribosomal RNA Modeling

 描述（由申请人提供）：该提案寻求通过冷冻电子显微镜和单粒子分类和重建方法，继续支持积极参与蛋白质合成的核糖体的结构和功能的探索。我们的显微镜配备的新的直接电子检测相机提供了前所未有的机会，以接近原子分辨率研究翻译的功能动力学。此外，我们已经实施并进一步开发的一种新能力，时间分辨cryo-EM，使我们能够研究反应时间在数十和数百毫秒内的过程，这些反应时间是解码，启动和错误检查机制研究中感兴趣的反应时间。与真细菌翻译领域的领先实验室合作，该提案的目的有三个：（1）研究新发现的追溯错误检查机制的结构基础，通过该机制，核糖体在多肽链上添加了错误的氨基酸后终止蛋白质合成;（2）以高分辨率可视化IF 2激活的两种状态下的前起始复合物，以表征伴随激活的结构变化，并开发激活过程的模型。在一个平行的研究中，我们将在几十到几百毫秒的范围内进行时间分辨实验，以跟踪亚基在起始过程中的连接过程。(3)通过从完整的翻译系统中收集大的单粒子数据集，我们将获得翻译的所有阶段的核糖体的详尽清单。使用基于数据相似性排序的流形嵌入新方法，与Abbas Ourmazd博士合作，我们将确定工作周期中构象状态的连续性，以及各种条件下核糖体的自由能景观。这些数据将使我们能够对细胞中最复杂的分子机器之一的结构动力学获得前所未有的见解。

 DESCRIPTION (provided by applicant): This proposal seeks continued support for exploration of structure and function of the ribosome actively engaged in protein synthesis, by means of cryo-electron microscopy and single-particle classification and reconstruction methods. The new direct electron detection camera our microscope is equipped with offers unprecedented opportunities to study the functional dynamics of translation at close to atomic resolution. Furthermore, a new capability we have implemented and further developed, time-resolved cryo-EM, allows us to investigate processes with reaction times in the tens and hundreds of milliseconds, which are reaction times of interest in the study of decoding, initiation and error check mechanisms. The aims of the proposal, in collaboration with leading labs in eubacterial translation, are threefold: (1) investigate the structural basis of a newly discovered retrospectiv error checking mechanism through which the ribosome terminates protein synthesis after an incorrect amino acid has been added to the polypeptide chain; (2) visualize a pre-initiation complex in two states of activation of IF2 at high resolution, to characterize the structural changes accompanying activation, and develop a model of the activation process. In a parallel study, we will perform time-resolved experiments in the range of tens to hundreds of millisecond, to follow the process of subunit joining during initiation. (3) By collecting large sigle particle datasets from complete translation systems, we will obtain an exhaustive inventory of ribosome in all phases of translation. Using a novel method of manifold embedding based on the similarity ordering of the data, in collaboration with Dr. Abbas Ourmazd, we will determine the continuum of conformational states during the work cycle, and the free energy landscape of the ribosome under various conditions. These data will allow us to gain unprecedented insights into the structural dynamics of one of the most complex molecular machines in the cell.