This process entails two specific transformations: one in the protein-bound state and the other in the solvent environment. In TI, the coupling parameter λ is essential for describing the step-by-step transition of the system's Hamiltonian from the starting ligand (λ=0) to the final ligand (λ=1). The free energy difference between the ligands is then computed by integrating the average values of ∂U (r N ;λ) ∂λ over the entire λ range. To estimate the RBFE, one must compute the difference in free energy variations between the protein-bound and solvent states: