Cryo-Tomography and Sub-Tomogram Averaging

  • Tomography integrates bidimensional projections into a 3D model.

  • From density maps to biological models
    Kudryashev et al., eLife 2013

  • Visualization of large data sets

  • Subtomogram averaging

  • Subtomogram classification
    Gil-Carton et al, Structure, 2015

 

Application: To obtain a 3D model for objects that are too thick for single particle electron microscopy (≥ 1 micrometer thick), e.g., small cells, cellular organelles, macromolecules and their complexes, viruses.

Method: Samples are vitrified by plunge freezing or by high-pressure freezing, cut into thin slices if necessary, and imaged at various tilt angles in the microscope using the lowest possible electron dose. Imaging under low dose conditions minimizes physical damage of the sample during data acquisition, but leads to extremely low signal-to-noise conditions. Sub-tomogram averaging recovers the signal by an intensive computational procedure, yielding structural resolutions of up to ~1 nm.

Advantages: Tomography generates three dimensional models of cellular samples by computational integration of different views, allowing the visualization of the proteins in a close-to-native environment.

Disadvantages: The sample can only be tilted by ± ~ 60° in the microscope. Thus, some 3D information is missing.

Sample requirements: