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: 10335173
• 负责人: JOACHIM FRANK
• 金额: $ 76.37万
• 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/10335173
• 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; 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， 翻译法规和翻译质量控制。为此，单粒子冷冻电子显微镜， PI实验室中率先开创的技术与世界专家有关细菌和细菌和 真核翻译。我们利用两种样品制备技术，标准和时间分辨 冷冻em。在低温EM的标准应用中，将样品移动到网格上，去除多余的液体 通过斑点，然后将网格浸入低温剂中。由于此过程需要几秒钟，所以 反应后无法捕获分子的短寿命（小于1000毫秒）。这 该实验室开发的替代技术是时间分辨的冷冻EM，从而通过混合开始反应 微流体芯片中的两个组件，使它们可以在定义的可变长度的通道中反应（10至10 1000毫秒），然后将反应产物喷射到网格上，而后者陷入了低温。在 这样，可以遵循反应的动力学，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：翻译 在大肠杆菌和酵母中启动，翻译终止，回收和质量控制，哺乳动物，EMCV病毒 接管宿主的核糖体。拟议研究的另一个目的是探索一种新方法 旨在生成来自庞大的连续体中存在的状态的低维状态图的数据分析 生物大分子的单粒子冷冻em图像的数据集。这样的映射可以用来 确定分子的自由能景观，其中包含有关功能相关的信息 构象轨迹。该方法将与领先的专家合作应用于两个 具有显着生物学和公共健康意义的膜相关分子：旋转ATPase和 囊性纤维化跨膜电导调节剂（CFTR）。