Structural Studies of Macromolecular Assemblies Using Cryo-EM

项目总结/摘要 拟议研究的主要目标是阐明翻译的基本过程， 翻译调控和翻译质量控制。为此，单粒子低温电子显微镜， 这项技术是PI实验室首创的，用于与世界各地的细菌和 真核生物翻译我们使用两种样品制备技术，标准和时间分辨 冷冻电镜在冷冻电镜的标准应用中，样品被移液到网格上，多余的液体被去除， 通过印迹，然后将网格插入冷冻剂中。由于此过程需要几秒钟，因此 不可能捕获反应后分子的短寿命（小于1000毫秒）状态。的 本实验室开发的另一种技术是时间分辨冷冻EM，通过混合 微流控芯片中的两种组分，允许它们在限定的、可变长度（10至20 μ m）的通道中反应。 1000 ms），然后在栅格投入冷冻剂时将反应产物喷射到栅格上。在 这样，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。这两种技术用于研究以下过程： 在E.大肠杆菌和酵母，哺乳动物翻译终止，再循环和质量控制，EMCV病毒 接管宿主的核糖体本研究的另一个目的是探索一种新的方法 数据分析的目的是从一个大的连续体中生成一个低维的状态图， 生物大分子的单粒子冷冻EM图像的数据集。这种映射可以用于 确定分子的自由能景观，包含与功能相关的信息 构象轨迹这种方法将应用于与领先的专家合作， 具有显著生物学和公共卫生意义的膜相关分子：旋转ATP酶和 囊性纤维化跨膜传导调节因子（Cystic Fibrosis trans-membrane conductance regulator，CFTR）

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).