膜蛋白（MPs）是所有生物膜的重要组成部分，有助于关键的蜂窝功能，包括信令，分子运输和能量代谢。因此,MPs是用于治疗发现的重要生物医学靶标。尽管低温电子显微镜的硬件和软件发展，以及MP样品制备，小于100kDa的MPs仍然难以进行结构研究。需要大量投资来克服低水平的天然丰富的蛋白质，MP疏水性以及构象和组成不稳定性。在这里，我们回顾了成功表达的样品制备方法，纯化并制备小的MPs，用于通过cryo-EM（总溶解分子量低于100kDa的那些）进行分析，以及检查数据处理的不同方法，并最终获得结构性解决方案。我们强调进程中每个阶段的共同挑战以及为克服这些问题而制定的战略。最后,我们讨论了通过冷冻EM研究亚100kDa膜蛋白的未来方向和机会。
Membrane proteins (MPs) are essential components of all biological membranes, contributing to key cellular functions that include signalling, molecular transport and energy metabolism. Consequently, MPs are important biomedical targets for therapeutics discovery. Despite hardware and software developments in cryo-electron microscopy, as well as MP sample preparation, MPs smaller than 100 kDa remain difficult to study structurally. Significant investment is required to overcome low levels of naturally abundant protein, MP hydrophobicity as well as conformational and compositional instability. Here we have reviewed the sample preparation approaches that have been taken to successfully express, purify and prepare small MPs for analysis by cryo-EM (those with a total solved molecular weight of under 100 kDa), as well as examining the differing approaches towards data processing and ultimately obtaining a structural solution. We highlight common challenges at each stage in the process as well as strategies that have been developed to overcome these issues. Finally, we discuss future directions and opportunities for the study of sub-100 kDa membrane proteins by cryo-EM.
