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. 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 Å (1.5 nm) resolution and confirm twofold symmetry. Using distance constraints generated by cross-linking mass 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.

径向辐条是控制真核纤毛运动的传感器装置中的关键元件。转导生物力学是纤毛生物学中长期存在的问题。径向辐条具有三个区域——辐条头、分叉颈部和辐条。尽管颈部和柄部不对称，但头部的双重对称性仍然存在争议。在这项工作中，我们使用单粒子冷冻电子显微镜 (cryo-EM) 分析以前所未有的分辨率生成整个径向辐条的 3D 结构。我们以 15 Å (1.5 nm) 分辨率显示头部区域并确认双重对称性。使用交联质谱产生的距离约束，我们在头部和颈部区域定位了两个成分，RSP2 和 RSP4。我们对分离的 RSP4、RSP9 和 RSP10 的生物物理分析证实了它们的寡聚状态。我们的结果使我们能够重新定义区域的边界，并提出径向辐条成分蛋白质的组织模型。