Summary of the image analysis and 3D image reconstruction procedure.

FSC profile indicating the resolution of ≈26 Å. Solid line in black is the FSC profile between two reconstructions calculated from two halves of the entire data set with 5-fold symmetry enforcement. Colored lines represent four FSC profiles between pairs of different azimuthal orientation at every 72° rotation for the reconstruction calculated without symmetry enforcement. These are also from one-half of the entire data set to be comparable with that of 5-fold averaging. The FSC curves fall off to 0.5 at 29 Å. Dotted and gray solid lines indicate the statistically expected error level in the FSC at and 5 σ, with and without 5-fold symmetry taken into account, respectively.

A model of the cap–filament complex built with the density map of the cap and an atomic model of the flagellar filament. (A) A model of the cap–filament complex built in five different side views, showing each of the five leg conformations and the gaps between the plate and the filament end. A front portion of the model is trimmed off to show the leg domains and gaps clearly. The cap plate is red, and the five leg domains are white, blue, green, brown, and purple. For the filament portion, the flagellin F41 subunits are represented as a space-filling model in yellow. (B) Pairwise comparison of the leg domain orientation in the HAP2 decamer (yellow) with those in the cap–filament model (other colors) to show the changes in their orientation on cap binding. (C) Pairwise comparison of leg domain orientation between neighboring ones in the cap–filament model to show changes in their orientation by one-step cap rotation. Number labels in B and C correspond to those in different views in A, whereas label 0 indicates the leg domain in the HAP2 decamer. Arrows in B and C indicate the direction of leg domain movement. (D and E) The movement of the cap on binding of a flagellin subunit (blue) before and after its binding, respectively. A thick cylindrical section covering a radial range containing domains D1 of flagellin and the leg domains of HAP2 is displayed in D and E. The changes in the leg domain orientation correspond to those in C. Domain colors are consistent in B–E. The figure was made with molscript (24), raster3d (25), volcube in situs (26), and our own in-house programs.

Electron cryomicrographs of the frozen hydrated HAP2 decamer complex and determination of its azimuthal orientation. (A) End-on views with a pentameric feature. (B) Side views with an elongated rectangular shape and thin plates at both ends. (Bar = 100 Å.) (C) Projection x profile for the determination of azimuthal orientation and 5-fold axis position of the cap. Six examples are shown. Thick lines represent those obtained from individual images, and thin lines represent those from the reference image. The determined azimuthal angles are 0°, 36°, 40°, 46°, 47°, and 57°. The two peaks in the density profile correspond to the leg domains on two sides of the particle and the trough to the central cavity. The interval of tick marks is 34.5 Å in the horizontal axis and an arbitrary unit in the vertical axis. These particular angles were chosen to demonstrate how this profile changes sensitively by a small change of angle.

Reconstructed 3D image of the HAP2 pentamer and decamer. Oblique view of the pentamer (A) and side view of the decamer (B), in a solid surface representation with 100% volume recovery are shown. The 5-fold symmetry is enforced. (Bar = 50 Å.) (C) Averaged end-on view. (D) Projection of the 3D reconstruction along the 5-fold axis. (E) Averaged side-view. (F) Pojection of the 3D reconstruction perpendicular to the 5-fold axis.