a, Conduits that extend through the PTCH1ECD in molA (right, yellow) and molB (left, cyan) identified in a simulation from Fig. 4d,e are shown as lines drawn at the central point of each tunnel overlaid with the starting structure. Exit points of each tunnel are noted. The midpoint traces of the tunnels are clustered as indicated by the colours. b, Model showing the consequences of cholesterol bound in the two different orientations for PTCH1 function. When cholesterol is bound in the hydroxyl-down orientation (left, cyan) in molB, the mouth of the SBD pocket is closed; however, conduits through the ECD, at the side of the ECD or just above the TMD are open for cholesterol. When the cholesterol attached to pShhNc (right, molA) is bound in the SBD, the mouth of the pocket is open to accommodate the ShhN protein chain, but the conduits through the PTCH1 ECD are closed, thereby blocking the cholesterol transporter activity of PTCH1. The palmitoyl group further inserts into and plugs the side exit in the ECD.