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

生命科学家的一个目标是了解细胞中的分子如何运作和相互作用。我们对分离的蛋白质结构域有很好的了解，我们可以用光学和电子显微镜看到细胞结构的整体组织。用分子显微镜技术获得的分辨率弥合了孤立域上的高分辨率信息与光学显微镜和常规电子显微镜技术给出的较低分辨率概览之间的差距。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，因为它们由具有不同功能的许多部分组成，并且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发挥作用的理解和洞察力。许多基本的细胞过程，如DNA转录，翻译和修复;或蛋白质合成，伴侣，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复合物的结构。然而，混合构象的存在可能是该技术的主要限制因素，因为通常假设所有分子或颗粒都是相同的。如果存在构象或结构的混合物，并且这些构象或结构未被检测和分离，则它们将被平均，并且可获得的分辨率将受到限制。面临的挑战是，如果构象物种可以被分离，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，该技术将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个优势，因为对多个状态的观察揭示了大分子复合物中的功能和机制。新的方法提供了一个客观的分析粒子的混合人口，从电子显微镜照片。它使状态的图像被分离，这样每个同质类都可以用标准方法处理。到目前为止，样品的不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。

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.