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

The radial spoke is a key element in a transducer apparatus controlling the motility of eukaryotic cilia. The transduction biomechanics is a long-standing question in cilia biology. The radial spoke has three regions - a spoke head, a bifurcated neck and a stalk. While the neck and the stalk are asymmetric, two-fold symmetry of the head has remained controversial.In this work we used single particle cryo-EM analysis to generate 3D structure of the whole radial spoke at unprecedented resolution. We show the head region at 15A resolution and confirm two-fold symmetry. Using distance constraints generated by crosslinking mass spectrometry we locate two components, RSP2 and 4 at the head/neck regions. Our biophysical analysis of isolated RSPs 4, 9, and 10 affirmed their oligomeric state. Our results enabled us to redefine the boundaries of the regions and propose a model of organization of the radial spoke component proteins.