Section: New Results

Single versus dual-axis cryo-electron tomography for reconstruction of microtubules assembled in vitro

Participant : Charles Kervrann.

Single-axis cryo-electron tomography of vitrified specimens has become a method of choice to reconstruct in three dimensions macromolecular assemblies in their cellular context or prepared from purified components. In [9] , we described a dual-axis acquisition scheme able to improve three-dimensional reconstructions of microtubules assembled in vitro. We showed that in single-axis tomograms, microtubules oriented close to the perpendicular of the tilt axis display diminished contrast, and ultimately transform into sets of parallel lines oriented in the direction of the electron beam when observed in cross-section. We analyzed projections in three-dimensional Fourier transform to demonstrate that imaging artifact is due to a decrease in the angular sampling of their equatorial components. Although the second orthogonal series of images does not fully complement the first one at the specimen level due to increased radiation damage, it still allows elongated features oriented in any directions to be correctly reconstructed, which might be essential for highly heterogeneous specimens such as cells.

Partners: Denis Chrétien, Audrey Guesdon and Sophie Blestel (UMR 6290 CNRS University of Rennes 1)