(A) A representative cross-section of a demembranated and negative-stained T. brucei flagellum, viewed from the proximal end (adapted from Hughes et al., 2012). Boxed region orients the view of the averaged 96-nm repeat along a DMT shown in B. (B) Cross-section view of the 96-nm repeat obtained by sub-tomogram averaging. Labeled are: the A- and B-tubule (At, Bt), Microtubule Inner Proteins (MIPs), Radial Spokes (RS), Inner Arm Dyneins (IAD), Nexin Dynein Regulatory Complex (NDRC), IAD-f-Intermediate Chain/Light Chain Complex (f-IC/LC), Outer Arm Dynein (OAD). The surface of the B-tubule from the adjacent DMT is visible on the right. The coloring scheme is as follows: cyan, OAD; red, IAD; blue, RS; green, NDRC; yellow, dynein f IC/LC. This scheme is consistently used throughout all main figures, figure supplements and videos unless stated otherwise. (C, D) Shaded surface rendering longitudinal views of the 96-nm repeat. Panel C shows the view from the center of the axoneme looking outward with the proximal end of the axoneme on the left and spoke heads removed for clarity (rotation relative to Panel D is shown). The surface of the B-tubule of the adjacent DMT is visible on top. Yellow and green arrows point to the inter-doublet connections formed by the f-connector and NDRC, respectively. Red arrows point to the proximal and distal holes in the inner junction between the A- and B-tubules. Panel D shows the view from the adjacent DMT, with proximal end of the axoneme on the left (rotation relative to panel B is shown). For reference, alpha (α) and beta (β) OAD are indicated, individual IADs and RS are labeled. (E) Shaded surface rendering of the averaged 96-nm repeat with the IAD, OAD and MIA complex removed, showing a massive structure at the base of the RS3 (see also Figure 3—figure supplement 1). Red arrows point to the density corresponding to the FAP59/172, 96-nm ruler (Oda et al., 2014a) between protofilaments A2 and A3. (F, G) Longitudinal (F) and transverse (G) density slices of the averaged 96-nm repeat. Red arrows in panels E and F point at the density of the FAP59/172 ruler between protofilaments A2 and A3. The red dashed line and perspective cartoon in panel F show the position and perspective of the cross-section shown in G, with the white arrow in panels F and G indicating the FAP59/172 ruler.