problems in the MALT1 system. A cumulative and cycle closure analysis suggested that a sampling time of 10 ns per λ window was adequate for this system. Simulations were mostly converged after 10 ns based on both the confidence interval of the RMSE getting within 0.7 kcal/mol and the overall low cycle closure errors. We analyzed the correlation and error statistics and the cycle closure at different amounts of sampling time to assess the convergence of the free energy estimate. As seen in Figure , the RMSE decreased with increasing simulation time until reaching a value of RMSE≈1.1 kcal/mol after around 80% sam-pling time, while the R 2 remained at a value around 0.85 after 30% sampling time. Since the RMSE stopped decreasing after 80% sampling time, we concluded that simulations had likely converged. Assessing how well ∆∆G values in a closed ligand cycle sum up to zero (cycle closure) is an additional metric for convergence. If the edges in a closed ligand cycle do not sum up to zero, simulations are not converged. However, if a cycle closes to zero, this does not necessarily mean that there are no sampling problems since there might be cancellation of errors. Here, we normalized the cycle closure to obtain an approximate contribution of a single edge to the cycle closure error: