Towards sub-millisecond cryo-EM grid preparation.
Towards sub-millisecond cryo-EM grid preparation.
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DOI：
10.1039/d2fd00079b
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发表时间：
2022-11-08
影响因子：
3.4
通讯作者：
Muench, Stephen P.
中科院分区：
文献类型：
--
作者：
Klebl, David P.;Kay, Robert W.;Sobott, Frank;Kapur, Nikil;Muench, Stephen P.
对于许多单颗粒冷冻电镜工作流程来说，样品制备仍然是一个重大问题，我们对该领域的理解和发展落后于图像处理和显微镜设计。在过去的几年中，越来越多的证据表明，样品制备过程中发生的许多问题都发生在样品驻留在传统印迹过程中产生的薄膜内的过程中。与此同时，针对时间分辨冷冻电镜实验开发了更快的网格准备方法，允许在毫秒时间尺度上捕获非平衡中间体。因此，一个重要的问题是我们能够以多快的速度准备适合冷冻电镜成像的网格，以及这在多大程度上缓解了样品制备中观察到的问题？在这里，我们使用一种专为时间分辨研究而开发的新颖方法，以估计亚 1 毫秒的时间尺度生成网格。虽然该方法有其自身的挑战，但用超快方法制备的脱铁铁蛋白的 3.8 Å 重建表明可以实现良好的分辨率。尽管我们使用核糖体样本显示比传统方法快几个数量级，但与空气-水界面的相互作用无法避免，且首选方向仍然存在。因此，这项工作表明可以捕获更快的反应，但提出了速度是否可以解决样品制备问题的问题。提出了一种新的冷冻电镜网格制备方法的第一个结果，该方法比以前的方法要快得多。短时间延迟有可能改善具有挑战性的样品的网格准备，并允许更快的时间分辨电磁。
Sample preparation is still a significant problem for many single particle cryo-EM workflows and our understanding and developments in the area lag behind that of image processing and microscope design. Over the last few years there has been growing evidence that many of the problems which occur during sample preparation are during the time the sample resides within the thin film created during the conventional blotting process. In parallel, faster grid preparation approaches have been developed for time-resolved cryo-EM experiments allowing for non-equilibrium intermediates to be captured on the ms timescale. Therefore, an important question is how fast can we prepare suitable grids for imaging by cryo-EM and how much does this mitigate the problems observed in sample preparation? Here we use a novel approach which has been developed for time-resolved studies to produce grids on an estimated sub-1 ms timescale. While the method comes with its own challenges, a 3.8 Å reconstruction of apoferritin prepared with the ultrafast method shows that good resolutions can be achieved. Although several orders of magnitude faster than conventional approaches we show using a ribosome sample, that interactions with the air–water interface cannot be avoided with preferred orientations still present. Therefore, the work shows that faster reactions can be captured but poses the question whether speed is the answer to problems with sample preparation. First results from a new cryo-EM grid preparation method are presented, significantly faster than previous methods. The short time delay has the potential to improve grid preparation for challenging samples and to allow faster time-resolved EM.
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DOI：
10.1107/s2059798321008810
发表时间：
2021-10-01
期刊：
Acta crystallographica. Section D, Structural biology
影响因子：
--
作者：
Klebl DP;White HD;Sobott F;Muench SP
通讯作者：
Muench SP
影响因子：
48
作者：
Zheng SQ;Palovcak E;Armache JP;Verba KA;Cheng Y;Agard DA
通讯作者：
Agard DA
DOI：
10.1107/s2059798320012474
发表时间：
2020-11-01
影响因子：
2.2
作者：
Tan, Yong Zi;Rubinstein, John L.
通讯作者：
Rubinstein, John L.
影响因子：
2.9
作者：
WHITE, HD;TAYLOR, EW
通讯作者：
TAYLOR, EW
DOI：
10.1107/s2059798318006551
发表时间：
2018-06-01
期刊：
Acta crystallographica. Section D, Structural biology
影响因子：
--
作者：
Afonine PV;Poon BK;Read RJ;Sobolev OV;Terwilliger TC;Urzhumtsev A;Adams PD
通讯作者：
Adams PD