On-grid and in-flow mixing for time-resolved cryo-EM.
On-grid and in-flow mixing for time-resolved cryo-EM.
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DOI：
10.1107/s2059798321008810
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发表时间：
2021-10-01
期刊：
影响因子：
--
通讯作者：
Muench SP
中科院分区：
文献类型：
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作者：
Klebl DP;White HD;Sobott F;Muench SP
时间分辨cryo-EM允许在毫秒时间尺度上的非平衡条件下研究蛋白质。在这里，在流和网格上的混合技术进行了直接比较，它被发现，网格上的反应可以通过空气-水的相互作用的影响，而在流反应由于层流反应时间的分布更广。时间分辨冷冻电子显微镜（TrEM）允许在毫秒时间尺度上的非平衡条件下研究蛋白质，允许分析大规模构象变化或组装和拆卸过程。然而，该技术正在发展中，很少与其他生化动力学研究进行比较。使用目前的方法，最短的时间延迟是毫秒时间尺度（105 -10 ms），由样品施加和玻璃化之间的延迟给出，并且产生更长的时间点需要额外的方法，例如在混合元件和喷嘴之间使用更长的延迟线，或者在网格上的孵育步骤。 为了比较方法，在混合和玻璃化之间延迟7-700 ms制备的网格上跟踪ATP与骨骼肌动球蛋白S1复合物的反应。 分类的cryo-EM数据允许动力学信息的推导，同意与以前的生化测量，显示快速解离，低占用率在稳态水解和重新绑定，一旦ATP已水解。然而，这种再结合的效果是不太明显时，网格上的混合使用，并可能受到影响的空气-水界面的相互作用。此外，由于层流剖面中的速度范围（时间扩展），特别是对于较长的时间延迟，流内混合导致反应时间的更宽分布。这项工作显示了TrEM的潜力，但也突出了进一步发展的挑战和机遇。
Time-resolved cryo-EM allows the study of proteins under non-equilibrium conditions on the millisecond timescale. Here, in-flow and on-grid mixing techniques are directly compared and it is found that on-grid reactions can be influenced by air–water interactions, whilst in-flow reactions give a broader distribution of reaction times due to laminar flow. Time-resolved cryo-electron microscopy (TrEM) allows the study of proteins under non-equilibrium conditions on the millisecond timescale, permitting the analysis of large-scale conformational changes or assembly and disassembly processes. However, the technique is developing and there have been few comparisons with other biochemical kinetic studies. Using current methods, the shortest time delay is on the millisecond timescale (∼5–10 ms), given by the delay between sample application and vitrification, and generating longer time points requires additional approaches such as using a longer delay line between the mixing element and nozzle, or an incubation step on the grid. To compare approaches, the reaction of ATP with the skeletal actomyosin S1 complex was followed on grids prepared with a 7–700 ms delay between mixing and vitrification. Classification of the cryo-EM data allows kinetic information to be derived which agrees with previous biochemical measurements, showing fast dissociation, low occupancy during steady-state hydrolysis and rebinding once ATP has been hydrolysed. However, this rebinding effect is much less pronounced when on-grid mixing is used and may be influenced by interactions with the air–water interface. Moreover, in-flow mixing results in a broader distribution of reaction times due to the range of velocities in a laminar flow profile (temporal spread), especially for longer time delays. This work shows the potential of TrEM, but also highlights challenges and opportunities for further development.