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首页> 外文期刊>Acta crystallographica. Section D, Structural biology >On-grid and in-flow mixing for time-resolved cryo-EM
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On-grid and in-flow mixing for time-resolved cryo-EM

机译：上网和子混合时间分辨cryo-EM

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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.
机译：时间分辨的低温电子显微镜(调节)允许下蛋白质的研究非平衡条件在毫秒时间表,允许大规模的分析或组装和构象变化拆卸过程。发展中国家和很少有比较与其他生化动力学研究。目前的方法,最短的时间延迟女士(~ 5 - 10毫秒级),给出的示例应用程序和之间的延迟玻璃化,生成时间点需要额外的使用等方法长之间的延迟线和混合元素喷嘴,或者一个孵化一步网格。比较方法,ATP的反应骨胳肌动球蛋白S1复杂之后网格的准备,中间有一个7 - 700毫秒的延迟混合和玻璃化。低温电子显微镜数据允许动态信息派生的同意以前的生化测量,显示快速分离、低在稳态水解和入住率重新连接一次ATP水解。这种重新绑定效果更明显当上网混合使用和可能的影响通过与空气的交互界面。此外,当在更广泛的混合结果反应时间的分布范围层流的速度剖面(时间传播),特别是长时间延迟。工作显示了这样的潜力,但也突出了挑战和机遇的进一步发展。

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