New methods for characterising heterogeneous populations of macromolecules using electron microscopy-defining function by direct imaging of states

使用电子显微镜表征大分子异质群体的新方法-通过状态直接成像定义功能

• 批准号: BB/H02414X/1
• 负责人: Alan Roseman
• 金额: $ 15.21万
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2011
• 资助国家: 英国
• 起止时间: 2011 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FH02414X%2F1
• 关键词: New; methods; characterising; heterogeneous; populations

A goal of life scientists is to understand how the molecules in cells operate and interact. We have a good understanding of isolated protein domains, and we can see the overall organisation of cell structure with light and electron microscopy. The resolution obtained with techniques in molecular microscopy bridges the gap between the high resolution information on isolated domains, and the lower resolution overview given by light microscopy and the conventional electron microscopy techniques. It is at the intermediate scale that the molecules are directly involved in regulating cellular processes. Biological macromolecular complexes are often molecular machines in that they consist of many parts with distinct functions, and movements or conformation changes are integral to their function and operation. Through observation of these complexes in their different states we could gain significantly improved understanding and insight on how these complexes work and function in the cell. Many fundamental cellular processes, such as DNA transcription, translation and repair; or protein synthesis, chaperoning, transport and degradation, are mediated by such large multi-component macromolecular complexes. With recent advances in electron microscopy instrumentation and analysis techniques we can examine the structures of these complexes in three dimensions at sub nanometre resolution. However the presence of mixed conformations can be a major limiting factor for this technique, as usually the assumption that all the molecules or particles are identical is applied. If a mixture of conformations or structures is present, and these are not detected and separated, then they will be averaged and the obtainable resolution will be limited. The challenge is that if the conformation species could be separated then invaluable information on function, dynamics, and mechanism could be obtained In this project we will develop a technique that will allow all the discrete states in the molecular population to be analysed, making the presence of heterogeneity an advantage, as the observation of multiple states reveals much about the function and mechanism in macromolecular complexes. The novel method provides an objective analysis of a mixed population of particles, from electron micrographs. It enables images of the states to be separated, so that each homogeneous class can be processed by the standard methods. Until now sample heterogeneity has been the biggest limiting factor for single particle analysis, but once this problem is solved it will become an advantage. Understanding the molecules involved in cellular processes enhances our knowledge of the mechanism of life, and provides a rational basis for the treatment of diseases.

生命科学家的一个目标是了解细胞中的分子是如何运作和相互作用的。我们对分离的蛋白质结构域有很好的了解，我们可以用光镜和电子显微镜看到细胞结构的整体组织。分子显微镜技术所获得的分辨率弥补了高分辨率分离域信息与光学显微镜和传统电子显微镜技术所提供的低分辨率概述之间的差距。在中间尺度上，分子直接参与调节细胞过程。生物大分子复合物通常是分子机器，因为它们由许多具有不同功能的部分组成，运动或构象变化是其功能和操作的组成部分。通过观察这些复合物的不同状态，我们可以大大提高对这些复合物如何在细胞中工作和功能的理解和见解。许多基本的细胞过程，如DNA转录、翻译和修复；或者蛋白质的合成、陪伴、运输和降解，都是由这种大的多组分大分子复合物介导的。随着电子显微镜仪器和分析技术的最新进展，我们可以在亚纳米分辨率的三维空间中检查这些配合物的结构。然而，混合构象的存在可能是该技术的主要限制因素，因为通常假设所有的分子或粒子都是相同的。如果存在混合构象或结构，而这些构象或结构没有被检测和分离，那么它们将被平均，可获得的分辨率将受到限制。挑战在于，如果可以分离构象物种，那么就可以获得关于功能，动力学和机制的宝贵信息。在这个项目中，我们将开发一种技术，允许分析分子群体中的所有离散状态，使异质性的存在成为一种优势，因为对多种状态的观察揭示了大分子复合物的功能和机制。新方法提供了一个客观的分析，从电子显微镜粒子的混合人口。它允许分离状态的图像，这样每个同构类都可以用标准方法进行处理。到目前为止，样品的非均匀性一直是单颗粒分析的最大限制因素，但一旦这个问题得到解决，它将成为一个优势。了解参与细胞过程的分子可以增强我们对生命机制的认识，并为疾病的治疗提供合理的基础。