Extended Data Fig. 5 |. Data collection and processing for isolated RS1.

a, Chromatogram showing the elution of RS1 from an anion-exchange column using a KCl gradient. The peak fraction containing RS1 is highlighted and elutes at ~0.7 M KCl. b, Silver-stained SDS-PAGE gel showing the purity of isolated RS1 following anion-exchange chromatography. The molecular weights of markers (in kDa) are indicated on the left. The result of mass spectrometry analysis of this sample is given in Supplementary Data 1. c, Section of a negative-stain electron micrograph showing homogeneous and monodisperse radial spokes. d, Selected two-dimensional class averages of particles selected from negative-stain electron micrographs. e, Section of an electron micrograph showing radial spokes in vitreous ice. Particles showing the characteristic T-shaped projection of radial spokes are circled. f, Selected two-dimensional class averages of radial spokes showing well defined spokeheads but nebulous density for the stalk consistent with flexibility at the neck. g, Schematic showing the processing of the isolated RS1 data. Following a consensus refinement, the spokehead and stalk were independently refined. The twofold rotational symmetry of the spokehead was exploited to improve the map quality. Further masked refinement was used to improve the flexible projections of the spokehead and the base and neck of the stalk. These individual maps were recombined to generate a final composite cryo-EM map. h, Angular distribution of the particle views used for the consensus reconstruction of isolated RS1. The height of the cylinders, colored from blue to red, represents the number of particles. The final density map of RS1 is shown in gray.