WaterKit protocol. The WaterKit simulation protocol is an iterative hydration process of a target structure, and can be summarized in four main steps (Fig. ): 1) identify and rank of all initial available hydrogen bond donor and acceptors (anchor points, AP) on the target structure; 2) for each anchor point a spherical model is used for the initial placement of individual water molecules, which are then converted to an explicit model to sample their orientation; this process is repeated until all available target anchor points are saturated (first hydration layer); 3) previously placed water molecules are then used to identify new anchor points to build multiple hydration layers, which are added until either the userdefined number of layers is placed, or there is no free volume left; 4) perform a quick energy minimization to relax proteinwater and water-water interactions. These steps produce a frame, and by repeating the process multiple times it is possible to generate an ensemble in which different water molecule configurations are sampled.