Visualization of macromolecular complexes using cryo-electron microscopy with FEI Tecnai transmission electron microscopes.
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).
Grassucci
Robert A
RA
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.
Taylor
Derek
D
Frank
Joachim
J
D020285:Cryoelectron Microscopy / Q000379:methods*; D007091:Image Processing, Computer-Assisted; D021621:Imaging, Three-Dimensional / Q000379:methods*; D046911:Macromolecular Substances / Q000032:analysis*; D046529:Microscopy, Electron, Transmission / Q000379:methods*
D046911:Macromolecular Substances