We performed 100 ns constant pH MD simulations at pH = pK a of systems ADA, AEA, AKA, AHA, AAA 1 , and AAA 2 (Table ). To enhance the sampling of the λ-coordinate in these systems, we ran the simulations without a barrier in the biasing potential (V bias (λ), Equation 5 in Aho et al. ). The correction potential (V MM (λ), Equation 5 in Aho et al. ) was obtained by fitting a third-order polynomial function to the ∂V /∂λ λ values of the reference trajectories. We will show later that for accurate and reproducible constant pH MD results, a higher-order fit is required. Nevertheless, in spite of its limited accuracy, using the same third-order fit for all system suffices to systematically compare the distributions of the relevant degrees of freedom and assess their convergence.