Scherer Sebastian, Kowal Julia, Chami Mohamed, Dandey Venkata, Arheit Marcel, Ringler Philippe, Stahlberg Henning
Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, CH-4058 Basel, Switzerland.
Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, CH-4058 Basel, Switzerland.
J Struct Biol. 2014 May;186(2):302-7. doi: 10.1016/j.jsb.2014.03.016. Epub 2014 Mar 28.
The introduction of direct electron detectors (DED) to cryo-electron microscopy has tremendously increased the signal-to-noise ratio (SNR) and quality of the recorded images. We discuss the optimal use of DEDs for cryo-electron crystallography, introduce a new automatic image processing pipeline, and demonstrate the vast improvement in the resolution achieved by the use of both together, especially for highly tilted samples. The new processing pipeline (now included in the software package 2dx) exploits the high SNR and frame readout frequency of DEDs to automatically correct for beam-induced sample movement, and reliably processes individual crystal images without human interaction as data are being acquired. A new graphical user interface (GUI) condenses all information required for quality assessment in one window, allowing the imaging conditions to be verified and adjusted during the data collection session. With this new pipeline an automatically generated unit cell projection map of each recorded 2D crystal is available less than 5 min after the image was recorded. The entire processing procedure yielded a three-dimensional reconstruction of the 2D-crystallized ion-channel membrane protein MloK1 with a much-improved resolution of 5Å in-plane and 7Å in the z-direction, within 2 days of data acquisition and simultaneous processing. The results obtained are superior to those delivered by conventional photographic film-based methodology of the same sample, and demonstrate the importance of drift-correction.
将直接电子探测器（DED）引入冷冻电子显微镜极大地提高了信噪比（SNR）和所记录图像的质量。我们讨论了DED在冷冻电子晶体学中的最佳使用方法，介绍了一种新的自动图像处理流程，并展示了将两者结合使用所实现的分辨率的大幅提高，特别是对于高度倾斜的样品。新的处理流程（现已包含在软件包2dx中）利用DED的高信噪比和帧读出频率来自动校正束流诱导的样品移动，并在采集数据时无需人工干预即可可靠地处理单个晶体图像。一个新的图形用户界面（GUI）将质量评估所需的所有信息浓缩在一个窗口中，允许在数据采集过程中验证和调整成像条件。使用这个新流程，在记录图像后不到5分钟即可获得每个记录的二维晶体的自动生成的晶胞投影图。在数据采集和同步处理的2天内，整个处理过程得到了二维结晶离子通道膜蛋白MloK1的三维重建，其平面分辨率提高到5Å，z方向分辨率提高到7Å。所获得的结果优于对同一样品采用传统基于摄影胶片的方法所得到的结果，并证明了漂移校正的重要性。
