Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323, USA.
Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323, USA.
J Struct Biol. 2021 Dec;213(4):107802. doi: 10.1016/j.jsb.2021.107802. Epub 2021 Oct 1.
While cryo-electron microscopy (cryo-EM) has revolutionized the structure determination of supramolecular protein complexes that are refractory to structure determination by X-ray crystallography, structure determination by cryo-EM can nonetheless be complicated by excessive conformational flexibility or structural heterogeneity resulting from weak or transient protein-protein association. Since such transient complexes are often critical for function, specialized approaches must be employed for the determination of meaningful structure-function relationships. Here, we outline examples in which transient protein-protein interactions have been visualized successfully by cryo-EM in the biosynthesis of fatty acids, polyketides, and terpenes. These studies demonstrate the utility of chemical crosslinking to stabilize transient protein-protein complexes for cryo-EM structural analysis, as well as the use of partial signal subtraction and localized reconstruction to extract useful structural information out of cryo-EM data collected from inherently dynamic systems. While these approaches do not always yield atomic resolution insights on protein-protein interactions, they nonetheless enable direct experimental observation of complexes in assembly-line biosynthesis that would otherwise be too fleeting for structural analysis.
虽然冷冻电子显微镜（cryo-EM）彻底改变了超分子蛋白质复合物的结构测定，使其能够抵抗 X 射线晶体学的结构测定，但 cryo-EM 的结构测定仍然可能受到过度构象灵活性或弱或瞬时蛋白质-蛋白质相互作用导致的结构异质性的影响。由于这种瞬时复合物对于功能通常至关重要，因此必须采用专门的方法来确定有意义的结构-功能关系。在这里，我们概述了在脂肪酸、聚酮和萜类化合物的生物合成中，cryo-EM 成功可视化瞬时蛋白质-蛋白质相互作用的例子。这些研究证明了化学交联用于稳定瞬时蛋白质-蛋白质复合物进行 cryo-EM 结构分析的实用性，以及使用部分信号减法和局部重建从固有动态系统收集的 cryo-EM 数据中提取有用的结构信息的用途。虽然这些方法并不总是能够提供关于蛋白质-蛋白质相互作用的原子分辨率的见解，但它们仍然能够直接观察到装配线生物合成中的复合物，否则这些复合物对于结构分析来说太短暂了。
