PDF(Pubmed)
Abstract：
The formation of a vitrified thin film embedded with randomly oriented macromolecules is an essential prerequisite for cryogenic sample electron microscopy. Most commonly, this is achieved using the plunge-freeze method first described nearly 40 years ago. Although this is a robust method, the behaviour of different macromolecules shows great variation upon freezing and often needs to be optimized to obtain an isotropic, high-resolution reconstruction. For a macromolecule in such a film, the probability of encountering the air-water interface in the time between blotting and freezing and adopting preferred orientations is very high. 3D reconstruction using preferentially oriented particles often leads to anisotropic and uninterpretable maps. Currently, there are no general solutions to this prevalent issue, but several approaches largely focusing on sample preparation with the use of additives and novel grid modifications have been attempted. In this study, the effect of physical and chemical factors on the orientations of macromolecules was investigated through an analysis of selected well studied macromolecules, and important parameters that determine the behaviour of proteins on cryo-EM grids were revealed. These insights highlight the nature of the interactions that cause preferred orientations and can be utilized to systematically address orientation bias for any given macromolecule and to provide a framework to design small-molecule additives to enhance sample stability and behaviour.
摘要：
嵌入随机取向的大分子的玻璃化薄膜的形成是低温样品电子显微镜的必要先决条件。最常见的是,这是使用近40年前首次描述的骤降冷冻方法实现的。虽然这是一个强大的方法，不同大分子的行为在冻结时显示出很大的变化，并且通常需要进行优化以获得各向同性，高分辨率重建。对于这种薄膜中的大分子，在印迹和冷冻之间的时间内遇到空气-水界面并采用首选方向的可能性非常高。使用优先定向粒子的3D重建通常会导致各向异性和不可解释的映射。目前,这个普遍的问题没有通用的解决方案，但是已经尝试了几种主要集中在使用添加剂和新型网格修饰的样品制备上的方法。在这项研究中,通过对选定的研究良好的大分子进行分析，研究了物理和化学因素对大分子取向的影响，并揭示了确定蛋白质在低温EM网格上行为的重要参数。这些见解突出了导致优选取向的相互作用的性质,并且可用于系统地解决任何给定大分子的取向偏差,并提供设计小分子添加剂以增强样品稳定性和行为的框架。
