Visualization of macromolecular complexes using cryo-electron microscopy with FEI Tecnai transmission electron microscopes

被引:28
作者
Grassucci, Robert A. [1 ]
Taylor, Derek [2 ]
Frank, Joachim [1 ,3 ]
机构
[1] Columbia Univ Coll Phys & Surg, Howard Hughes Med Inst, Dept Biochem & Mol Biophys, New York, NY 10032 USA
[2] Wadsworth Ctr, Hlth Res Inc, Albany, NY 12201 USA
[3] Columbia Univ, Dept Biol Sci, New York, NY 10027 USA
关键词
D O I
10.1038/nprot.2007.474
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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).
引用
收藏
页码:330 / 339
页数:10
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