Layer-line patterns of Class I and Class II filaments and 3D maps reveal typical actin repeating structures. (A) Typical Class I (top) and Class II (bottom) lumenal filaments are extracted as 3D sub-volumes from the tomographic reconstruction and shown as projected in Z. The corresponding layer-line patterns are shown beside the projection. The characteristic 4-nm tubulin layer line was visible in all patterns. Parameters measured for more filaments are provided in Table S1. Class I filaments exhibited a clear reflection at 5.94 ± 0.03 nm, which is the pitch of the left-handed genetic helix of actin (Actin Turn [L]) and a reflection at 29.53 ± 0.81 nm corresponding to the crossover spacing of the right-handed long pitch helices (Actin Cross) as measured in the individual images. Reflections were observed in the Class II filaments at similar positions to Class I 6.11 ± 0.09 nm and 27.44 ± 2.28 nm, with an additional strong meridional reflection at 6.17 ± 0.06 nm (errors are SD). SEM is provided in Table S1 with measurements of additional actin reflection (Actin Turn [R]) at the pitch of the right-hand genetic helix. (B) 3D maps from representative extracted filament volumes using real space helical reconstructions with helical parameters derived from the models presented in Fig. S3 and Table S1. Maps are low pass filtered to 30 Å.