Chou Hui-Ting, Evans James E, Stahlberg Henning
Molecular & Cellular Biology, University of California, Davis, CA, USA.
Methods Mol Biol. 2007;369:331-43. doi: 10.1007/978-1-59745-294-6_16.
Electron crystallography studies the structure of two-dimensional crystals of membrane proteins or other crystalline arrays. This method has been used to determine the atomic structures of six membrane proteins and tubulin, as well as several other structures at a slightly lower resolution, where secondary structure motifs could be identified. To preserve the high-resolution structure of 2D crystals, the meticulous sample preparation for electron crystallography is of outmost importance. Charge-induced specimen drift and lack of specimen flatness can severely affect the resolution of images for tilted samples. However, sample preparations that sandwich the two-dimensional crystals between symmetrical carbon films reduce the charge-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 cryoelectron microscope using either the imaging or the electron diffraction mode has to be performed after low-dose procedures. Spot scanning further reduces the charge-induced specimen drift.
电子晶体学研究膜蛋白或其他晶体阵列的二维晶体结构。该方法已用于确定六种膜蛋白和微管蛋白的原子结构，以及其他几种分辨率稍低的结构，在这些结构中可以识别二级结构基序。为了保留二维晶体的高分辨率结构，电子晶体学细致的样品制备至关重要。电荷诱导的样品漂移和样品平整度不足会严重影响倾斜样品图像的分辨率。然而，将二维晶体夹在对称碳膜之间的样品制备方法可减少电荷诱导的样品漂移，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。在低温电子显微镜中使用成像或电子衍射模式进行数据收集必须在低剂量程序之后进行。点扫描进一步减少电荷诱导的样品漂移。
