Extended Data Fig. 3: Sample preparation and cryo-EM data processing part I (using cryoSPARC) to determine the 3D structure of the DMT and RS3 in mouse respiratory cilia.

a) Samples for cryo-EM single particle analysis were prepared by excising the trachea from WT mice, using calcium-shock and detergent treatment to remove the ciliary axonemes from the multiciliated respiratory epithelium, and splaying the axonemes into DMTs using ATP and mild salt-treatment. The DMTs were then plunge-frozen, cryo-EM images were recorded in a semi-automated fashion on a Titan Krios, and after image processing (see b) the 3D structure of the DMT repeat was reconstructed. shows the image processing steps (part I) for cryo-EM single particle analysis using the software cryoSPARC⁹⁹. After DMT signal subtraction, the micrographs were importing into Relion¹⁰² for the 3D reconstruction of RS3 (see image processing part II, Extended Data Fig. 4). c) Example of a motion-corrected cryo-EM micrograph of a DMT with regularly spaced RS3 (arrowheads), the characteristic spacing allowed manual particle picking of RS3; attempts of automatic and AI-assisted particle picking failed. This experiment was repeated 50,000 times independently with similar results. d) Examples of 2D class averages centered on the docking site of RS3 to the DMT. e, f) Cross-sectional (left) and longitudinal (right) slices (e) and isosurface renderings (f) of the 3D reconstructed DMT with local resolution distribution indicated as color-gradient (estimated by cryoSPARC⁹⁹). Details of the DMT are well-resolved, such as the tektin-MIPs in the A-tubule (arrowhead in e). However, the alignment is dominated by the DMT and the RS3 structure is blurred (arrows in e). Scale bars: (a, c) 50 nm, (d, e) 20 nm.