The structure and symmetry of the radial spoke protein complex in Chlamydomonas flagella

The radial spoke is a key element in a transducer apparatus controlling themotility of eukaryotic cilia. The transduction biomechanics is a longstanding question in cilia biology. The radial spoke has three regions - a spoke head, a bifurcated neck and a stalk. Although the neck and the stalk are asymmetric, twofold symmetry of the head has remained controversial. In this work we used single particle cryo-electron microscopy (cryo-EM) analysis to generate a 3D structure of the whole radial spoke at unprecedented resolution. We show the head region at 15 A (1.5 nm) resolution and confirmtwofold symmetry. Using distance constraints generated by cross-linkingmass spectrometry, we locate two components, RSP2 and RSP4, at the head and neck regions. Our biophysical analysis of isolated RSP4, RSP9, and RSP10 affirmed their oligomeric state. Our results enable us to redefine the boundaries of the regions and propose a model of organization of the radial spoke component proteins.