Structural Studies of Macromolecular Assemblies Using Cryo-EM

使用冷冻电镜进行大分子组装体的结构研究

• 批准号: 10552673
• 负责人: JOACHIM FRANK
• 金额: $ 76.78万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-01 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10552673
• 关键词: ATP phosphohydrolase; Behavior; Biological; Collaborations; Cryoelectron Microscopy; Cystic Fibrosis Transmembrane Conductance Regulator; Data Analyses; Dimensions; Encephalomyocarditis virus; Escherichia coli; Free Energy; Image; Kinetics; Length; Liquid substance; Maps; Membrane; Methods; Microfluidic Microchips; Molecular Conformation; Molecular Structure; Preparation; Procedures; Process; Protein Biosynthesis; Public Health; Quality Control; Reaction; Recycling; Research; Resolution; Ribosomes; Sampling; Specialist; Structure; Techniques; Time; Translation Initiation; Translation Process; Translational Regulation; Translations; Virus; Visualization; Yeasts; large datasets; macromolecular assembly; macromolecule; millisecond; novel; particle

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).

项目概要/摘要 拟议研究的主要目标是阐明翻译的基本过程， 翻译监管和翻译质量控制。为此，单粒子冷冻电子显微镜， 该技术是 PI 实验室首创的，与世界细菌和细菌专家合作使用 真核翻译。我们利用两种样品制备技术：标准技术和时间分辨技术 冷冻电镜。在冷冻电镜的标准应用中，将样品移至网格上，去除多余的液体 通过吸干，然后将网格浸入冷冻剂中。由于此过程需要几秒钟，因此 不可能捕获反应后分子的短暂（小于 1000 毫秒）状态。这 该实验室开发的替代技术是时间分辨冷冻电镜，通过混合开始反应 微流控芯片中的两个组件，使它们能够在定义的可变长度（10 至 1000 ms），然后将反应产物喷射到网格上，同时网格投入冷冻剂中。在 这样，可以跟踪反应动力学，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： 翻译 大肠杆菌和酵母中的起始、哺乳动物中的翻译终止、回收和质量控制、EMCV 病毒 接管宿主的核糖体。拟议研究的另一个目标是探索一种新方法 数据分析的目的是从大数据中生成连续体中存在的状态的低维地图 生物大分子的单粒子冷冻电镜图像数据集。这样的映射可用于 确定分子的自由能景观，包含与功能相关的信息 构象轨迹。该方法将与领先专家合作应用于两个 具有突出生物学和公共卫生意义的膜相关分子：旋转 ATP 酶和 囊性纤维化跨膜电导调节器（CFTR）。