The PDF file includes:

• Fig. S1. Purification of the hedgehog ligand and PTCH1.
• Fig. S2. Cryo-EM and single-particle analysis workflow.
• Fig. S3. FSC plots and 3D reconstruction details.
• Fig. S4. Features of the map and the atomic model of the protein.
• Fig. S5. Disease-linked mutations in PTCH1 are present within the SSD and SSDL regions of the protein.
• Fig. S6. Mutations designed to disrupt sterol binding sites in PTCH1 fail to suppress hedgehog pathway activation.
• Table S1. Cryo-EM data collection, single-particle analysis, and model building statistics.

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Other Supplementary Material for this manuscript includes the following:

• Movie S1 (.mp4 format). The density elements corresponding to bound sterols in the PTCH1 TM-focused map.