使用FEI Tecnai透射电子显微镜通过冷冻电子显微镜对大分子复合物进行可视化。
Visualization of macromolecular complexes using cryo-electron microscopy with FEI Tecnai transmission electron microscopes.
作者信息
Grassucci Robert A, Taylor Derek, Frank Joachim
机构信息
Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University College of Physicians and Surgeons, 630 West 168th Street, New York, New York 10032, USA.
出版信息
Nat Protoc. 2008;3(2):330-9. doi: 10.1038/nprot.2007.474.
Abstract
This protocol details the steps used for visualizing the frozen-hydrated grids as prepared following the accompanying protocol entitled 'Preparation of macromolecular complexes for visualization using cryo-electron microscopy.' This protocol describes how to transfer the grid to the microscope using a standard cryo-transfer holder or, alternatively, using a cryo-cartridge loading system, and how to collect low-dose data using an FEI Tecnai transmission electron microscope. This protocol also summarizes and compares the various options that are available in data collection for three-dimensional (3D) single-particle reconstruction. These options include microscope settings, choice of detectors and data collection strategies both in situations where a 3D reference is available and in the absence of such a reference (random-conical and common lines).
摘要
本方案详细介绍了用于观察冷冻水合网格的步骤，该网格是按照随附的题为“使用冷冻电子显微镜观察大分子复合物的制备”的方案制备的。本方案描述了如何使用标准的冷冻转移支架或使用冷冻盒加载系统将网格转移到显微镜上，以及如何使用FEI Tecnai透射电子显微镜收集低剂量数据。本方案还总结并比较了用于三维（3D）单颗粒重建的数据采集中可用的各种选项。这些选项包括显微镜设置、探测器的选择以及在有3D参考和没有3D参考（随机锥形和公共线）的情况下的数据收集策略。
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