Need for Speed: Examining Protein Behavior during CryoEM Grid Preparation at Different Timescales.
Need for Speed: Examining Protein Behavior during CryoEM Grid Preparation at Different Timescales.
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DOI：
10.1016/j.str.2020.07.018
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发表时间：
2020-11-03
期刊：
影响因子：
--
通讯作者：
Muench SP
中科院分区：
文献类型：
--
作者：
Klebl DP;Gravett MSC;Kontziampasis D;Wright DJ;Bon RS;Monteiro DCF;Trebbin M;Sobott F;White HD;Darrow MC;Thompson RF;Muench SP
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。颗粒分区到空气-水界面，即使在快速（ms）网格制作更高的网格制作速度可以导致更大的角展度的标本更高的网格制作速度可以减少颗粒损伤和亚基解离不同的网格制作设备可以影响样品的浓度在冰这里，我们利用新的网格制作程序，以研究在cryoEM网格生产速度对蛋白质颗粒行为的影响。这些结果使我们能够更好地了解不同的网格制作方法如何影响所得的网格相对于颗粒损伤，优选的取向，和所得的浓度。
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. Particles partition to the air-water-interface even during fast (ms) grid making Higher grid-making speeds can result in greater angular spread of the specimen Higher grid-making speeds may reduce particle damage and subunit dissociation Different grid-making apparatus can affect sample concentration in the ice Here, we take advantage of new grid-making procedures to investigate the effect of speed in cryoEM grid production on protein particle behavior. These results allow us to better understand how different grid-making approaches can affect the resultant grid with respect to particle damage, preferred orientation, and resultant concentration.
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