Assembly intermediates of orthoreovirus captured in the cell

Fig. 1. FIB milling and tomograms. a SEM image of vitrified infected cells on EM grid prior to FIB milling. Scale bar 1 mm. b SEM image after FIB milling. White arrow shows one of the four-milled lamellae. Scale bar 100 μm. c, d Example tomogram of particles within cell (d shows a close up view). Scale bars 3000 and 1800 Å, respectively. e–g Representatives of stars, empty, and full virion-like particles, respectively taken from SIRT reconstruction by Tomo3D44. All three images are at the same scale and the scale bar corresponds to 450 Å. These images are representative of three lamella from which eight tomographic series were collected.

Fig. 2. Sub-tomogram averaged reconstructions. a–c Overviews of sub-tomogram averaging of stars, empty- and full virion-like particles respectively using IMOD/PEET32. Scale bar length 500 Å. d Cross-section through the fivefold of map (b). Circled in red is density attributed to the polymerase. The σ1 spike protrudes from the surface (a σ1 trimer is fitted). e Surface rendering of the emClarity map for the empty virion-like particle coloured by radius (scale bar as for panel h), a σ3/μ1 heterohexamer is circled in red. f, g Fits of the models (see “Methods”) into the empty virion-like particle pseudo sixfold emClarity map, σ3 in purple, μ1 in cyan. h, i emClarity SLP map. h Coloured by radius. i Fit of λ1 subunits into the density, molecules A and B coloured green and red respectively. j Slice through the superposed sub-tomogram averaged emClarity maps; empty virion-like particle in blue and SLP in orange. The data were taken from five tomographic series (selected from 8 in total). Information on the number of particles used is given in Supplementary Table 1.

Fig. 3. Rearrangement between SLP and virion-like particles. a, b Side views of the conformational changes within the λ1 subunits between virion-like particle (blue) and the SLP where the two portions are coloured raspberry and forest, the latter portion being closest to the fivefold. c Interface areas between λ1 molecules in the SLP. Highlighted in yellow is the area shown in (d) with the arrow indicating the viewpoint. d Close up view of the ratchet of the AB interface. The A molecules are on the left. SLP molecules A and B coloured grey and orange, respectively. Molecules A and B for the virion-like particle are coloured pink and green, respectively. To show the ratchet more clearly the A molecules of each AB pair have been superposed. For the B molecules ratchetting helices and the pivot helix are coloured equivalently for the SLP and the virus (the pivot helix is shown in purple). Arrows show the rotation between the SLP and virus. e SLP density (PEET map) unaccounted for by λ1. Within the icosahedral asymmetric unit there are two distinct sets of density, one close to the twofold is coloured yellow, the other close to the threefold is coloured magenta. f, g close up of the…

Fig. 4. Similarity between the SLP and the indented form of Phi8 procapsid. The structures are coloured according to the distance of equivalent Cα’s between the indented and expanded forms of each virus. For clarity, only the indented state of each virus is shown. For both viruses molecule A moves more than molecule B, although it should be noted that there is greater movement between the two particle forms in Phi8.