Need for speed: Examining protein behaviour during cryoEM grid preparation at different timescales
Need for speed: Examining protein behaviour during cryoEM grid preparation at different timescales
复制标题
对速度的需求：在不同时间尺度的冷冻电镜网格制备过程中检查蛋白质行为
DOI：
10.1101/2020.05.14.095372
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发表时间：
2020
期刊：
影响因子：
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通讯作者：
Klebl D
中科院分区：
文献类型：
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作者：
Klebl D
许多新技术正在开发中，以提高低温电子显微镜（cryoEM）网格制备的质量和再现性。在这里，我们系统地研究了三种大分子复合物的制备，使用三种不同的玻璃化设备（Vitrobot，变色龙，和时间分辨cryoEM设备）在不同的时间尺度上，包括在6 ms内（迄今为止报道的最快）的网格，以询问不同时间点的空气-水界面处的颗粒行为。结果表明，不同的大分子复合物可以以高度可变的方式响应cryoEM样品制备过程中形成的薄膜环境，从而阐明cryoEM样品制备难以优化的原因。我们证明，减少样品应用和玻璃化之间的时间只是一个工具，以提高cryoEM网格质量，但它不太可能是一个通用的“银”，以提高每个cryoEM样品制备的质量。
A host of new technologies are under development to improve the quality and reproducibility of cryoelectron microscopy (cryoEM) grid preparation. Here we have systematically investigated the preparation of three macromolecular complexes using three different vitrification devices (Vitrobot, chameleon, and a time-resolved cryoEM device) on various timescales, including grids made within 6 ms (the fastest reported to date), to interrogate particle behavior at the air-water interface for different timepoints. Results demonstrate that different macromolecular complexes can respond to the thin-film environment formed during cryoEM sample preparation in highly variable ways, shedding light on why cryoEM sample preparation can be difficult to optimize. We demonstrate that reducing time between sample application and vitrification is just one tool to improve cryoEM grid quality, but that it is unlikely to be a generic "silver bullet" for improving the quality of every cryoEM sample preparation.