Extended Data Fig. 1: Sample preparation and processing strategies used to resolve the 96-nm repeat of axonemal doublet microtubules (DMTs) from different mammalian motile cilia.

(a) Stitched low-magnification micrographs of a disintegrated bovine sperm cell (left) and corresponding high-magnification image of a DMT with bound radial spokes (RSs) (right). A total of 45,431 such micrographs were processed for this study. (b) Schematic of the isolation of human and porcine oviduct cilia using a syringe inserted into the oviduct. Human cilia were splayed into individual DMTs after demembranating and incubating with ATP to promote DMT-DMT sliding. A similar workflow was used to prepare DMTs from bovine oviducts and porcine brain ventricles. (c) General processing scheme used for single particle analysis (SPA) of DMTs from bovine sperm, bovine/human oviductal cilia, and porcine brain ventricle cilia. All steps were performed in cryoSPARC unless otherwise stated. See Methods and Supplementary Figs. 1–7 for details. (d) Cryo-EM map of the bovine sperm 96-nm repeat with local resolution ranges for individual axonemal complexes indicated. (e) Porcine oviduct cilia subtomogram averaging (STA) workflow. See Methods for details. (f) Fourier shell correlation (FSC) curves for the four segments used to reconstruct the porcine DMT at binning factor 2 by STA. (g) Comparison of oviduct cilia reconstructions obtained by SPA and STA.