neutralize ensures a net-neutral charge in the simulation system. First, it adds ions to make the system neutral for the configured initial state. It can also add ions at a given concentration if specified by the user. Second, neutralize adds buffer particles, which will compensate for charge fluctuations during constant pH simulations. By default, neutralize will add as many buffer particles as the total number of titratable sites N sites in the system. However, the number of buffers may also manually be set by the user. The general recommendation is that the λ-value of the buffer should be close to 0.5 (corresponds to zero charge for the buffers) during the entire simulation. While this can be achieved by increasing the total number of buffers to up to twice the number of titratable sites, we nevertheless recommended to use smaller buffer concentrations in order to not significantly alter the solvent properties. Thus, the optimal buffer concentration can be selected in the range from 2 √ πN sites , average charge fluctuation, First, the group name is provided inside the square brackets, followed by alternative names, which will be used to search for residues that phbuilder should make titratable.