Implementation of a cryo-electron tomography tilt-scheme optimized for high resolution subtomogram averaging.
Cryo-electron tomography (cryoET) allows 3D structural information to be obtained from cells and other biological samples in their close-to-native state. In combination with subtomogram averaging, detailed structures of repeating features can be resolved. CryoET data is collected as a series of images of the sample from different tilt angles; this is performed by physically rotating the sample in the microscope between each image. The angles at which the images are collected, and the order in which they are collected, together are called the tilt-scheme. Here we describe a "dose-symmetric tilt-scheme" that begins at low tilt and then alternates between increasingly positive and negative tilts. This tilt-scheme maximizes the amount of high-resolution information maintained in the tomogram for subsequent subtomogram averaging, and may also be advantageous for other applications. We describe implementation of the tilt-scheme in combination with further data-collection refinements including setting thresholds on acceptable drift and improving focus accuracy. Requirements for microscope set-up are introduced, and a macro is provided which automates the application of the tilt-scheme within SerialEM.
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, Germany.
Wim J H
WJH
Hagen
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, Germany.
William
W
Wan
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, Germany. Electronic address: john.briggs@embl.de.
John A G
JAG
Briggs
D000465:Algorithms; D020285:Cryoelectron Microscopy / Q000379:methods*; D055032:Electron Microscope Tomography / Q000379:methods*; D007091:Image Processing, Computer-Assisted / Q000379:methods; D021621:Imaging, Three-Dimensional / Q000379:methods
Cryo-electron microscopy; Electron tomography; Subtomogram averaging; Tilt-scheme