A total of 7628 cryo-EM movies were motion corrected using MotionCor2 (40). Contrast transfer function (CTF) was estimated using Gctf on non–dose-weighted aligned images (41); micrographs with estimated resolution exceeding 4 Å were discarded, leaving 3260 micrographs for downstream processing. The PTCH1Δ-ShhNC24II particles were autopicked in relion-2.1.0 using templates generated by 2D classification of 1306 manually picked particles (42), resulting in a selection of 707,620 particles. After several rounds of 2D classification, 430,811 particles were selected for 3D classification. The procedure for 3D classification with four classes was performed in relion-2.1.0, using a 3D model of PTCH1Δ-apo low pass filtered to 30 Å. The initial model was generated de novo in cisTEM using a dataset composed of 165,533 PTCH1Δ-apo particles selected after 2D classification in relion-2.0. The best 3D class of the PTCH1Δ-ShhNC24II complex consisted of 200,679 particles, showed clear secondary structure elements (α helices), and featured a bound molecule of the hedgehog ligand. This class was selected for 3D refinement. Refinement of this class gave a map with a resolution of 3.6 Å [based on Fourier shell correlation (FSC) at 0.143]. Reprocessing of this class using relion-3.0, including motion correction, CTF refinement, and Bayesian particle polishing (43), resulted in a new map with a resolution of 3.5 Å (“F”). Further refinement was performed using the mask that excluded the detergent micelle, continuing from the last iteration. This resulted in a map at 3.4-Å resolution. The same procedure was repeated substituting the mask with a detergent and ectodomain-free mask, resulting in a 3.3-Å resolution map after postprocessing. Postprocessing was performed in relion-3.0 using a B factor of −50 Å2. The processing steps and map improvements are illustrated in figs. S2 and S3.

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