Extended Data Fig. 9: Cryo-EM data and image processing flow for ShhN–DISP1-A complex, and comparison to Drosophila HhNDisp complex.
From: Dispatched uses Na+ flux to power release of lipid-modified Hedgehog
a, A representative cryo-EM micrograph (n = 1687) and highly populated reference-free 2D class averages for ShhN–DISP1-A. b, Schematic flow-chart illustrating the image processing used for ShhN–DISP1-A data. Thumbnail images are shown for reference-based 3D classes and high resolution refinements. Dashed black boxes indicate 3D classes selected for the next processing step, with class particle counts in red and refinement GS-FSC resolutions in black. The label, ‘3D classification with references’ indicates that explicit ‘apo’ and ‘complex’references were used to seed the classification. c, Left, the cryo-EM density from our ShhN–DISP1-A complex (transparent grey), overlaid on a ribbon diagram of ShhN (goldenrod). Right, cryo-EM density from the Drosophila HhNDisp complex in Cannac et al24. (transparent grey, EMD: 10464), overlaid on the ribbon diagram of HhN (orchid, model extracted from PDB ID: 6TD6). Middle, superimposition of the ShhN–DISP1-A and HhNDisp complex models reported here and in Cannac et al24., with mouse DISP1-A and ShhN in khaki and goldenrod, and Drosophila Disp and HhN in pink and orchid. The model of Drosophila HhN was based on docking of a Drosophila HhN structure56 within a 4.8 Å density map. Relative to Drosophila HhN, murine ShhN is translated upwards, away from the membrane, and rotated towards the right. Asterisks indicate corresponding positions near the N termini of ShhN and HhN proteins. This difference is somewhat puzzling in light of the ability of mammalian DISP1 to rescue Drosophila disp mutant function6, and we cannot definitively account for it. One functional difference is that mammalian DISP1 cooperates with SCUBE2 for its Hedgehog-releasing activity, whereas Drosophila lacks a Scube orthologue. The ECD domain split and the Hedgehog interaction with the furin-cleaved linker arm together help explain the requirement for furin cleavage22 in DISP1 function.
