Goldie Kenneth N, Abeyrathne Priyanka, Kebbel Fabian, Chami Mohamed, Ringler Philippe, Stahlberg Henning
Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University Basel, Basel, Switzerland.
Methods Mol Biol. 2014;1117:325-41. doi: 10.1007/978-1-62703-776-1_15.
Electron crystallography is used to study membrane proteins in the form of planar, two-dimensional (2D) crystals, or other crystalline arrays such as tubular crystals. This method has been used to determine the atomic resolution structures of bacteriorhodopsin, tubulin, aquaporins, and several other membrane proteins. In addition, a large number of membrane protein structures were studied at a slightly lower resolution, whereby at least secondary structure motifs could be identified.In order to conserve the structural details of delicate crystalline arrays, cryo-electron microscopy (cryo-EM) allows imaging and/or electron diffraction of membrane proteins in their close-to-native state within a lipid bilayer membrane.To achieve ultimate high-resolution structural information of 2D crystals, meticulous sample preparation for electron crystallography is of outmost importance. Beam-induced specimen drift and lack of specimen flatness can severely affect the attainable resolution of images for tilted samples. Sample preparations that sandwich the 2D crystals between symmetrical carbon films reduce the beam-induced specimen drift, and the flatness of the preparations can be optimized by the choice of the grid material and the preparation protocol.Data collection in the cryo-electron microscope using either the imaging or the electron diffraction mode has to be performed applying low-dose procedures. Spot-scanning further reduces the effects of beam-induced drift. Data collection using automated acquisition schemes, along with improved and user-friendlier data processing software, is increasingly being used and is likely to bring the technique to a wider user base.
电子晶体学用于研究呈平面二维（2D）晶体形式的膜蛋白，或其他晶体阵列，如管状晶体。该方法已被用于确定细菌视紫红质、微管蛋白、水通道蛋白及其他几种膜蛋白的原子分辨率结构。此外，还以略低的分辨率研究了大量膜蛋白结构，借此至少可以识别二级结构基序。为了保留精细晶体阵列的结构细节，冷冻电子显微镜（cryo-EM）可对脂质双分子层膜中接近天然状态的膜蛋白进行成像和/或电子衍射。为了获得二维晶体的最终高分辨率结构信息，精心进行电子晶体学样品制备至关重要。电子束引起的样品漂移和样品平整度不足会严重影响倾斜样品图像可达到的分辨率。将二维晶体夹在对称碳膜之间的样品制备方法可减少电子束引起的样品漂移，并且可以通过选择网格材料和制备方案来优化制备的平整度。在冷冻电子显微镜中使用成像或电子衍射模式进行数据采集时，必须采用低剂量程序。逐点扫描进一步减少了电子束引起的漂移影响。使用自动采集方案以及改进的、用户友好的数据处理软件进行数据采集的情况越来越普遍，这可能会使该技术拥有更广泛的用户群体。
