Structural Studies of Macromolecular Assemblies Using Cryo-EM

项目摘要/摘要 拟议研究的主要目的是阐明翻译的基本过程， 翻译监管和翻译质量控制。为此，单粒子低温电子显微镜， 这项由国际和平研究所实验室首创的技术，被用于与世界各地的细菌和 真核翻译。我们使用了两种样品制备技术，标准样品和时间分辨样品 冷冻机。在低温电磁的标准应用中，样品被输送到栅格上，多余的液体被去除 通过印迹，然后网格被插入到制冷剂中。由于此过程需要几秒钟，因此 不可能捕捉反应后分子的短暂状态(小于1000毫秒)。这个 这个实验室开发的另一种技术是时间分辨低温电磁，它通过混合来启动反应 微流控芯片中的两种成分，允许它们在定义的、可变长度的通道中进行反应(10到 1000ms)，然后将反应产物喷洒到栅格上，当后者进入制冷剂时。在……里面 通过这种方式，可以跟踪反应的动力学，同时可以捕获中间状态 并以高分辨率可视化。这两种技术被用来研究以下过程： 哺乳动物EMCV病毒在大肠杆菌和酵母中的引发、翻译终止、回收和质量控制 接管宿主的核糖体。拟议研究的另一个目标是探索一种新的方法 数据分析的一种方法，它试图从大量的数据中生成存在于连续统中的状态的低维地图 生物大分子的单粒子低温电子显微镜图像的数据集。这样的映射可用于 确定分子的自由能图景，包含与功能相关的信息 构象轨迹。该方法将应用于与两位顶尖专家的合作 具有显著生物学和公共卫生意义的膜相关分子：旋转ATPase和 囊性纤维化跨膜电导调节剂。

Project Summary/Abstract The main objective of the proposed studies is the elucidation of fundamental processes of translation, translational regulation and translational quality control. To this end, single-particle cryo-electron microscopy, the technique pioneered in the PI's lab, is used in collaborations with world specialists on bacterial and eukaryotic translation. We make use of two techniques of sample preparation, standard and time-resolved cryo-EM. In the standard application of cryo-EM, samples are pipetted onto the grid, excess liquid is removed by blotting, and the grid is then plunged into the cryogen. Since this procedure requires several seconds, it is not possible to capture short-lived (less than 1000 millisecond) states of a molecule following a reaction. The alternative technique developed in this lab is time-resolved cryo-EM, whereby a reaction is started by mixing two components in a microfluidic chip, allowing them to react in a channel of defined, variable length (10 to 1000 ms), and then spraying the reaction products onto the grid as the latter in plunged into the cryogen. In this way, the kinetics of a reaction can be followed and, at the same time, intermediate states can be captured and visualized at high resolution. These two techniques are used to study the following processes: translation initiation in E. coli and yeast, translation termination, recycling and quality control in mammalian, EMCV virus takeover of the host's ribosome. Another objective of the proposed studies is the exploration of a novel method of data analysis that seeks to generate a low-dimensional map of states existing in a continuum from a large dataset of single-particle cryo-EM images of a biological macromolecule. Such a mapping can be used to determine the free-energy landscape of the molecule, containing information on the function-related conformational trajectories. This method will be applied in collaborations with leading experts to two membrane-associated molecules with eminent biological and public health significance: rotary ATPase and Cystic Fibrosis trans-membrane conductance regulator (CFTR).