The radial spoke (RS) transmits mechanochemical signals from the central pair apparatus (CP) to axonemal dynein arms to coordinate ciliary motility. The RS head, directly contacting with CP, differs dramatically in morphology between protozoan and mammal. Here we show the murine RS head is compositionally distinct from the Chlamydomonas one. Our reconstituted murine RS head core complex consists of Rsph1, Rsph3b, Rsph4a, and Rsph9, lacking Rsph6a whose orthologue exists in the Chlamydomonas RS head. We present the unprecedented cryo-EM structure of RS head core complex at 4.5 angstrom resolution and identified the subunit location and their interaction network. In this complex, Rsph3b, Rsph4a, and Rsph9 forms a compact body with Rsph4a serving possibly as an assembly scaffold and Rsph3b in a location that might link the head with stalk. Interestingly, two Rsph1 subunits constitute the two stretching-arms possibly for optimized RS-CP interaction. We also propose a sawtooth model for the RS-CP interaction. Our study suggests that the RS head experiences profound remodeling to probably comply with both structural and functional alterations of the axoneme during evolution.

Distinct architecture and composition of mouse axonemal radial spoke head revealed by cryo-EM