Although high‐resolution single‐particle cryo‐electron microscopy (cryo‐EM) is now producing a rapid stream of breakthroughs in structural biology, it nevertheless remains the case that the preparation of suitable frozen‐hydrated samples on electron microscopy grids is often quite challenging. Purified samples that are intact and structurally homogeneous – while still in the test tube – may not necessarily survive the standard methods of making extremely thin, aqueous films on grids. As a result, it is often necessary to try a variety of experimental conditions before finally finding an approach that is optimal for the specimen at hand. Here, we summarize some of our collective experiences to date in optimizing sample preparation, in the hope that doing so will be useful to others, especially those new to the field. We also hope that an open discussion of these common challenges will encourage the development of more generally applicable methodology. Our collective experiences span a diverse range of biochemical samples and most of the commonly used variations in how grids are currently prepared. Unfortunately, none of the currently used optimization methods can be said, in advance, to be the one that ultimately will work when a project first begins. Nevertheless, there are some preferred first steps to explore when facing specific problems that can be more generally recommended, based on our experience and that of many others in the cryo‐EM field.

中文翻译：

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优化单颗粒冷冻电镜“标准”样品制备的当前结果


尽管高分辨率单颗粒冷冻电子显微镜（cryo-EM）现在在结构生物学领域取得了一系列快速突破，但在电子显微镜网格上制备合适的冷冻水合样品通常仍然相当具有挑战性。 。完整且结构均匀的纯化样品（尽管仍在试管中）可能不一定能够通过在网格上制作极薄水膜的标准方法而幸存下来。因此，在最终找到最适合当前样本的方法之前，通常需要尝试各种实验条件。在这里，我们总结了迄今为止在优化样品制备方面的一些集体经验，希望这对其他人，特别是对该领域的新手有用。我们还希望对这些共同挑战的公开讨论将鼓励开发更普遍适用的方法。我们的集体经验涵盖了各种生化样品以及目前网格制备方式中最常用的变化。不幸的是，目前使用的优化方法都不能提前说是在项目刚开始时最终起作用的方法。尽管如此，根据我们和冷冻电镜领域许多其他人的经验，在面临特定问题时，可以更普遍地推荐一些首选的第一步。