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).

项目总结/文摘