Single particle and molecular assembly analysis of polyribosomes by single- and double-tilt cryo electron tomography

Ultramicroscopy. 2013 Mar;126:33-9. doi: 10.1016/j.ultramic.2012.12.009. Epub 2012 Dec 20.


Cryo electron tomography (cryo-ET) can provide cellular and molecular structural information on various biological samples. However, the detailed interpretation of tomograms reconstructed from single-tilt data tends to suffer from low signal-to-noise ratio and artefacts caused by some systematically missing angular views. While these can be overcome by sub-tomogram averaging, they remain limiting for the analysis of unique structures. Double-tilt ET can improve the tomogram quality by acquiring a second tilt series after an in-plane rotation, but its usage is not widespread yet because it is considered technically demanding and it is rarely used under cryo conditions. Here we show that double-tilt cryo-ET improves the quality of 3D reconstructions so significantly that even single particle analysis can be envisaged despite of the intrinsically low image contrast obtained from frozen-hydrated specimens. This is illustrated by the analysis of eukaryotic polyribosomes in which individual ribosomes were reconstructed using single-tilt, partial and full double-tilt geometries. The improved tomograms favour the faster convergence of iterative sub-tomogram averaging and allow a better 3D classification using multivariate statistical analysis. Our study of single particles and molecular assemblies within polysomes illustrates that the dual-axis approach is particularly useful for cryo applications of ET, both for unique objects and for structures that can be classified and averaged.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cryoelectron Microscopy / methods*
  • Image Processing, Computer-Assisted
  • Imaging, Three-Dimensional / methods
  • Multivariate Analysis
  • Polyribosomes / chemistry*
  • Signal-To-Noise Ratio
  • Tomography / methods*