The crystal structures and unbinding trajectories were visually inspected to investigate why the agonist and cationic antagonists employed one single type of path to leave the binding site. In the crystal structure of the active TLR8 (PDB ID: 3W3L), the agonist 1 forms hydrogen bonds with the side chain of residue Asp543* (with the asterisk denoting chain B) and with the backbone of Thr574*. Residues Tyr353 and Asp545* are located close to the agonist 1, sterically preventing it from exiting the binding site through the external path, while residues Arg429 and Tyr567* sterically prevent ligand 1 from exiting through the internal path (Figure ). The rotation of Tyr353 in the trajectories created space for ligand 1 motion, serving as a precursor for ligand 1 to exit the binding site through the external path (Figure ). Comparison between the initial and final frames of the τRAMD trajectories shows that there is a shift in the chi angles displayed by Tyr353 in the final frames (Figure ), further supporting the importance of the rotation of Tyr353 for ligand unbinding. Taken together, these results suggest that the unbinding of agonist 1 via the external path is facilitated by rotation of Tyr353, explaining the preference of agonist 1 for this path.