As mentioned, phbuilder can also convert existing simulations to constant pH simulations. To demonstrate this feature, we set up constant pH simulations based on an existing cardiotoxin V system and from a membrane protein that was prepared using CHARMM-GUI. We first set up a run starting from the existing cardiotoxin V system and checked whether this produced similar results to the constant pH simulation set up from the initial protein. The evolution of protonation states and protein structures for the prepared cardiotoxin V system are shown in supplementary figure . Protonation states evolved together with the atomic coordinates and shared similar trends with the simulations of cardiotoxin prepared from the initial protein structure (Figure ).