High-speed high-resolution data collection on a 200 keV cryo-TEM.
High-speed high-resolution data collection on a 200 keV cryo-TEM.
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DOI：
10.1107/s2052252522000069
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发表时间：
2022-03-01
期刊：
影响因子：
3.9
通讯作者：
Strauss JD
中科院分区：
文献类型：
--
作者：
Peck JV;Fay JF;Strauss JD
本研究探讨了高速，高分辨率，单粒子数据收集使用200千电子伏的塔罗斯北极配备了加坦K3 DED的参数。 近原子分辨率的电子显微镜图是从以每小时720部电影的速度收集的数据集中获得的。成功的单粒子低温电子显微镜（cryo-EM）项目的局限性包括稳定的样品生成，生产质量cryo-EM网格与随机取向的颗粒嵌入在薄玻璃冰和获得显微镜时间。为了解决显微镜时间的限制，测试了在200 keV Talos Arctica低温透射电子显微镜上以每小时720部电影的速度更有效地收集数据的方法。 在这项研究中，探索了提高数据收集速度的关键参数，包括：（1）使用光束图像移位方法在每个载物台位置采集多个图像，（2）采用孔间距为R0.6/1的UltrAufoil TEM网格，（3）收集硬件分箱数据，（4）调整SerialEM中的图像移位延迟因子。在这里，在分析中获得了8个分辨率为1.8-1.9 μ m的小鼠脱铁蛋白的EM图，每个数据集的数据收集时间从56 min到2 h不等。 以每小时500个电影的速度从过夜数据收集中获得小鼠脱铁铁蛋白在1.78 nm处的EM图，并进行亚组分析，通过图像移位距离和图像移位延迟未观察到数据质量的显著变化。本文详述的发现和操作程序允许单颗粒冷冻EM结构测定的快速周转。
This study explores parameters for high-speed, high-resolution, single-particle data collection using a 200 keV Talos Arctica equipped with a Gatan K3 DED. Near-atomic resolution electron microscopy maps were obtained from datasets collected as fast as 720 movies per hour. Limitations to successful single-particle cryo-electron microscopy (cryo-EM) projects include stable sample generation, production of quality cryo-EM grids with randomly oriented particles embedded in thin vitreous ice and access to microscope time. To address the limitation of microscope time, methodologies to more efficiently collect data on a 200 keV Talos Arctica cryo-transmission electron microscope at speeds as fast as 720 movies per hour (∼17 000 per day) were tested. In this study, key parameters were explored to increase data collection speed including: (1) using the beam-image shift method to acquire multiple images per stage position, (2) employing UltrAufoil TEM grids with R0.6/1 hole spacing, (3) collecting hardware-binned data and (4) adjusting the image shift delay factor in SerialEM. Here, eight EM maps of mouse apoferritin at 1.8–1.9 Å resolution were obtained in the analysis with data collection times for each dataset ranging from 56 min to 2 h. An EM map of mouse apoferritin at 1.78 Å was obtained from an overnight data collection at a speed of 500 movies per hour and subgroup analysis performed, with no significant variation observed in data quality by image shift distance and image shift delay. The findings and operating procedures detailed herein allow for rapid turnover of single-particle cryo-EM structure determination.