a, Section of an electron micrograph showing radial spokes (marked with an asterisk) bound to a doublet microtubule in vitreous ice. b, Processing scheme used to generate reconstructions of mechanoregulatory complexes bound to doublet microtubules (DMT, gray). To resolve various structural features with 96-nm periodicity (RS1 spokehead/stalk, RS2 spokehead/stalk, RSP1-RSP1 interface, RS3S, IDAc, or N-DRC baseplate/lobe), it was necessary to use a combination of tubulin signal subtraction (TSS), shifting the center (SC) of coordinates to the feature of interest, focused refinement (FR) and 3D classification without alignment (3C). When possible, the box size was reduced (RB) to 256 or 384 instead of 512 pixels to facilitate data processing. c, Angular distribution of the particle views used for reconstruction of on-doublet RS2. Similar distributions were obtained for on-doublet RS1. The height of the cylinders, colored from blue to red, represents the number of particles. The final density map of RS2 is shown in gray. d, Superimposition of the on-doublet RS1 and RS2 spokeheads confirms that they have identical structure.