破译大分子复合物中的蛋白质-配体相互作用对于理解分子机制至关重要,潜在的生物过程,和药物开发。近年来,低温样品电子显微镜(cryoEM)已成为一种强大的技术,可以确定大分子的结构并研究以近原子分辨率结合配体的方式。由于跨感兴趣分子的各向异性分辨率和数据中的固有噪声,在冷冻EM图中识别和建模非蛋白质分子通常是具有挑战性的。在这篇文章中,向读者介绍了目前用于配体鉴定的各种软件和方法,模型建筑,和使用选定的大分子细化原子坐标。识别配体存在的最简单方法之一,正如烯醇酶所说明的,是减去有和没有配体获得的两个图谱。即使在较高的阈值下,配体的额外密度也可能在差异图中突出。有实例,如在代谢型谷氨酸受体mGlu5的情况下所示,当不能生成这样的简单差异图。最近引入的衍生Fo-Fc省略图的方法可以用作验证和证明配体存在的工具。最后,以研究良好的β-半乳糖苷酶为例,分析了分辨率对冷冻EM图中配体和溶剂分子建模的影响,并对冷冻EM如何用于药物发现进行了展望。
Deciphering the protein-ligand interactions in a macromolecular complex is crucial for understanding the molecular mechanism, underlying biological processes, and drug development. In recent years, cryogenic sample electron microscopy (cryoEM) has emerged as a powerful technique to determine the structures of macromolecules and to investigate the mode of ligand binding at near-atomic resolution. Identifying and modeling non-protein molecules in cryoEM maps is often challenging due to anisotropic resolution across the molecule of interest and inherent noise in the data. In this article, the readers are introduced to various software and methods currently used for ligand identification, model building, and refinement of atomic coordinates using selected macromolecules. One of the simplest ways to identify the presence of a ligand, as illustrated with the enolase enzyme, is to subtract the two maps obtained with and without the ligand. The extra density of the ligand is likely to stand out in the difference map even at a higher threshold. There are instances, as shown in the case of metabotropic Glutamate receptor mGlu5, when such simple difference maps cannot be generated. The recently introduced method of deriving the Fo-Fc omit map can serve as a tool for validating and demonstrating the presence of the ligand. Finally, using the well-studied β-galactosidase as an example, the effect of resolution on modeling the ligands and solvent molecules in cryoEM maps is analyzed, and an outlook on how cryoEM can be used in drug discovery is presented.