Extended Data Fig. 10: Conformational dynamics link intramembrane Na⁺ site occupancy to Hedgehog release.

a, Three-dimensional variability analysis (3DVA) of the ShhN–DISP1-A complex dataset reveals a conformational series with ShhN bound or absent at opposite ends of the first principal component (PC1). Front (first line) and top (second line) views of reconstructed densities from the extremes of PC1 (unbound, khaki; bound, salmon) are shown, with a superimposed view of these extremes in the centre. The overall changes in ECD position are illustrated by lines drawn atop the reconstructed densities and by schematized diagrams to the left. Reconstructed densities at the Na⁺ coordination sites in the transmembrane domain indicate that these sites change from fully occupied in the unbound state to site I only occupied in the bound state. See also Supplementary Video 4. b, 3DVA analysis of the apoprotein preparation shows a similar conformational series along PC1, along with similar shifts in Na⁺ site occupancy.