4.6A Cryo-EM reconstruction of tobacco mosaic virus from images recorded at 300 keV on a 4k x 4k CCD camera

Abstract

Tobacco mosaic virus (TMV) is a plant virus with a highly ordered organisation and has been described in three different structural states: As stacked disks without RNA (X-ray crystallography), as a helical form with RNA (X-ray fibre diffraction) and as a second distinct helical form with RNA (cryo-EM). Here we present a structural analysis of TMV as a test object to assess the quality of cryo-EM images recorded at 300 keV on a CCD camera. The 4.6A TMV structure obtained is consistent with the previous cryo-EM structure and confirms that there is a second helical form of TMV. The structure here also shows that with a similar number of TMV segments an equivalent resolution can be achieved with a 4k CCD camera at 300 keV.

Fig. 1

Image processing scheme. A schematic of the image processing steps used to reconstruct the helical form of TMV.

Fig. 2

Helical reconstruction of TMV. The fully corrected sharpened reconstruction from 260 000 ASU of TMV, shown from the side (a), as a central axial-section (b), and in cross-section. The docked atomic coordinates of the 2OM3 structure are shown in cartoon representation (rainbow coloured).

Fig. 3

The refined atomic coordinates of a TMV monomer fitted into the map. Side view (a) and top view (b) of the HR region of the refined monomer fitted into the density map of TMV contoured at 2σ. Side view (c) of the LR region of the refined monomer fitted into the density map of TMV contoured at 1σ. The LR region was contoured at a lower threshold as the inner channel-lining loop disappears at higher thresholds, probably due to greater conformational flexibility.