We used the Major Urinary Protein (MUP) structure with the PDB ID 1ZNK. The system was constructed using the TIP3P water model, the ff14SB force field for the protein, and GAFF for the ligand. The protein-ligand complex was solvated in rectangular boxes using tleap with initial buffer sizes of 11 Å. The net charge of the system was neutralized by addition of Na + . The simulations were performed on GPUs with AMBER20's GPUaccelerated PMEMD simulation code. During equilibration, Cartesian restraints to the starting structure were applied to all ligand and protein heavy atoms, with a force constant of 5 kcal mol -1 Å -2 . After a brief minimization (50 steps of steepest descent plus 450 steps of conjugate gradient), the system was sequentially heated at a fixed volume with linear ramps joining the temperatures 5, 100, 200, and 298.15 K, at 20 ps per ramp, with each ramp followed by an additional 20 ps with pressure coupling. All simulations used a Langevin integrator with a 2 fs timestep with a friction coefficient of 2 ps -1 . Heating steps were run in NVT, while equilibration and production steps were run in NPT with pressure regulated at 1 atm with a Monte Carlo barostat. The SHAKE algorithm was used to constrain hydrogen bond lengths. The standard AMBER protocol for non-bonded interactions was followed. Thus, the particle mesh Ewald (PME) method was used for periodic boundary conditions with an 8 Å cutoff for the short-ranged PME contribution as well as LJ interactions. A long-range continuum correction was used for the dispersive term. Cartesian restraints with a force constant of 5 kcal mol -1 Å -2 were applied to the Cα atoms and ligand atoms during the production run.