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

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.

► Double-tilt cryo-ET improves 3D reconstructions thus making single particle analysis possible.
► Dual-axis cryo-ET data favour a faster convergence of iterative sub-tomogram averaging.
► Individual ribosomes were reconstructed from single-tilt, partial/ full double-tilt geometries.
► Double-tilt cryo-ET facilitates analysis of larger molecular assemblies such as in cell sections.
► Dual-axis cryo-ET is applicable to unique objects and to structures that can be classified and averaged.