Even though AFE calculations and MD/NCMC may have similar computational cost, at least in the case of few binding modes, AFE calculations are not always that straightforward to implement. For example, the core region of the ligands might require additional restraints to prevent them from drifting out of the binding pocket during the intermediate alchemical states. Additionally, rotatable bonds might need harmonic restraints such that they sample only a specific binding modes. Furthermore, it is hard to converge AFE calculations to a reasonable accuracy of 1.0 kcal/mol. In practice, AFE calculations are popularly coupled with tempering methods, such as REST/REST2, for better sampling and convergence. Unlike AFE calculations, with MD/NCMC we have not used any restraints with the ligand having full freedom to move around in the binding pocket if it wants to do so and were able to achieve a satisfactory level of convergence. 4.6. Umbrella sampling requires more computation to converge to the correct population. As an additional point of comparison, we ran umbrella sampling simulations with ligand 1 to compare the computational cost with MD/NCMC simulations. Unlike MD/NCMC simulations, where we were able to get converged simulations easily (or by running longer for larger ligands), we had to optimize and tune the umbrella sampling protocol a number of times to achieve a converged population distribution for the two binding modes (Figure and).