Mouse radial spoke 3 is a metabolic and regulatory hub in cilia.

Cilia are microtubule-based organelles that have important roles in cell sensing, signaling and motility. Recent studies have revealed the atomic structures of many multicomponent ciliary complexes, elucidating their mechanisms of action. However, little is known about the structure, proteome and function of full-length radial spoke 3 (RS3), a conserved complex that transmits mechanochemical signals to coordinate ciliary motility. Here, we combined single-particle cryo-electron microscopy, cryo-electron tomography, proteomic analysis and computational modeling to determine the three-dimensional structure and atomic model of RS3 from mouse respiratory cilia. We reveal all RS3 components, including regulatory and metabolic enzymes such as a protein kinase A subunit, adenylate kinases (AKs) and malate dehydrogenases. Furthermore, we confirm RS3 loss in AK7-deficient mice, which exhibit motility defects. Our findings identify RS3 as an important regulatory and metabolic hub that maintains sufficient adenosine triphosphate for sustained ciliary beating, providing insights into the etiology of ciliopathies.