(a) left, Comparison of the Fen49 computational model (green, grey side-chains, yellow fentanyl) with the apo Fen49 crystal structure (light blue, purple side-chains). For clarity, the molecule of PEG observed in the crystal structure has been omitted. Computationally designed side-chains, plus Y80, are shown as sticks. Fentanyl-associated water molecules are shown as red spheres and connected with yellow dashed lines. (a) right, Fen49 model aligned with the Fen49*-fentanyl complex crystal structure (wheat, magenta side-chains, orange fentanyl). The fentanyl tertiary amine-associated chloride and carbonyl water are shown as green and red spheres, respectively. (b) left, The Fen49*-complex binding site, colored as in Figure 3a. Side-chains involved in binding fentanyl are shown, with the corresponding 2Fo-Fc electron density from the refined maps, contoured at 1.0 σ (Figure 3—figure supplement 8). (b) right, Ligplot (Laskowski and Swindells, 2011) 2D representation of all residues contacting fentanyl. Two superimposed layers are shown, keeping Y80 and Y166 fixed for orientation. Residues making hydrophobic and polar contacts are labeled with black and green font, respectively. Water molecules and the chloride ion are shown as blue circles and green circles, respectively. Green dashed lines represent hydrogen bonds (distances in Å). A second molecule of fentanyl was observed on the periphery of the binding cavity (Figure 3—figure supplement 9).