RBFE calculations for PLpro. For each mutation, three 10-15 ns production simulations were performed at each λ-window (see Fig. for details on extension of initial 10 ns trajectory). The gradients were extracted and analyzed with three different equilibration methods: AED (see Approach), a 2 ns equilibration period, and a 5 ns equilibration period. The equilibrated gradients were then decorrelated and averages were extracted. 10 short-run simulations were performed with Protocols A, B, C, and D for each mutation (see Table for protocol details).