6.2. Ways to test whether a target is within the domain of applicability of the BFE approach. In general, free energy calculations require a particular target to be within the domain of applicability of a method, but it is often difficult to know a priori what that domain of applicability is. Thus, when starting to work on a new system, it can make sense to first test convergence of the free energy estimate on a smaller subset to assess whether a new target is within the domain of applicability of the method. By domain of applicability we here mean that the binding mode is known and that there are no slow binding mode changes as ligands are swapped, e.g. slow water rearrangement or slow protein conformational changes. Convergence of the free energy estimate can be tested with different methods. For example, running calculations in replicates and looking at the standard deviation across repeats, as well as running transformations in opposite directions (A → B and B → A) can help assess convergence. Additionally, a high cycle closure in a cycle of ligand transformations is another indicator that an important degree of freedom was not sampled sufficiently. In some cases more ambitious convergence tests may be beneficial, such as starting simulations from different available protein structures (e.g. structures that had been crystallized with different ligands bound) as well as starting simulations with dif-ferent water rearrangements in the binding site. If the starting structures impact the calculated binding free energy, convergence has not been achieved yet.