National Centre for Biological Sciences, Tata Institute of Fundamental Research.
Institute for Stem Cell Science and Regenerative Medicine;
J Vis Exp. 2024 Jul 19(209). doi: 10.3791/66310.
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.
解析大分子复合物中的蛋白-配体相互作用对于理解分子机制、潜在的生物过程和药物开发至关重要。近年来，低温冷冻样品电子显微镜（cryoEM）已成为确定大分子结构以及研究配体结合模式的强大技术，可以达到近原子分辨率。由于感兴趣的分子的各向异性分辨率和数据固有的噪声，在 cryoEM 图谱中识别和建模非蛋白分子通常具有挑战性。本文向读者介绍了目前用于配体鉴定、模型构建和使用选定大分子的原子坐标精修的各种软件和方法。鉴定配体存在的最简单方法之一，如图醇酶(enolase)所示，是减去有配体和没有配体时获得的两个图谱。即使在更高的阈值下，配体的额外密度也很可能在差异图谱中突出显示。如图氨酸代谢型谷氨酸受体 mGlu5 的情况所示，有时无法生成这种简单的差异图谱。最近引入的 Fo-Fc 省略映射推导方法可以作为验证和证明配体存在的工具。最后，使用研究充分的β-半乳糖苷酶（β-galactosidase）作为示例，分析了分辨率对 cryoEM 图谱中配体和溶剂分子建模的影响，并介绍了 cryoEM 在药物发现中的应用前景。
