The next simulations for replica equilibration and ligand unbinding were carried out with GROMACS-RAMD version 2.0 . Each of the five replicas underwent an additional equilibration with an NVT ensemble with the Berendsen thermostat 56 and a constant temperature of 300 K. The equilibration consisted of 10000000 steps with a time step of 0.002 ps, resulting in 20 ns simulation time. Electrostatic forces were calculated using the PME method. This resulted in five equilibrated replicas, each serving as a starting point for 20 individual tauRAMD simulations for ligand unbinding, resulting in 100 unbinding events for each complex. The tauRAMD simulations were performed with an NPT ensemble, the Nose-Hoover thermostat with a constant temperature of 313 K and the Parrinello-Rahman barostat with a constant pressure of 1 bar. The maximum duration of the simulations was 50000000 steps, with a time step of 0.002 ps, resulting in a maximum simulation time of 100 ns. The Van-der-Waals forces were computed using a cutoff of 1.2 nm. Electrostatic forces were calculated using the PME method with a realspace cutoff of 1.2 nm, PME order of four and a Fourier grid spacing of 1.2 Å. The linear center of mass motion of the protein and ligand was removed every 100 steps of the simulation. The covalent bonds to hydrogens in the solute were constrained using the LINCS algorithm. For the tauRAMD parameters, the magnitude of the force applied to the ligands during the simulations was 9 kcal/molÅ, the threshold distance was set to 0.0025 nm and the evaluation frequency was set to 50 fs. The reference atom of the protein was the backbone amine of G351. An unbinding event was identified when the ligand showed a distance above 6 nm from the reference atom.