Three-dimensional reconstruction
Three-dimensional reconstructions using SPIDER were carried out on the best classes, those showing the clearest protofilament substructure, using helical symmetry.56 Two parameters had to be determined for the reconstruction: the cross-over repeat (Fig. 1a) and the subunit repeat (Fig. 1d). The cross-over repeat was determined by selecting a small region around one cross-over from the class average and by using cross correlation to determine the position of the other cross-over. The cross-over length was checked visually on the class average. Determination of the subunit repeat was more difficult, and several methods were used to verify the results. The starting point was the 5.88-nm repeat observed in diffraction patterns of negative stain images (Fig. 1e). Then, each map was reconstructed with a range of subunit repeats (4.5–7 nm), and the maps were examined to find the ones with the most distinct features, the highest contrast, and the best-matching reprojections (Supplementary Fig. 4). Based on these comparisons, a subunit repeat of 5.78 nm was used for the A-type maps, and a subunit repeat of 5.25 nm was used for the B-type maps. Short segments were reconstructed, and these were helically averaged to generate a full repeat so that reprojections could be compared to the class averages.