(A) Schematic illustration of two enhanced sampling methods, that both alter the energy landscape to force the simulation out of energy minima; accelerated MD (aMD) and metadynamics (MetaD), see the main text. (B) Simple potential of mean force (PMF) calculation. The system is sampled along a reaction coordinate, typically the membrane–protein distance, and the force necessary to keep the protein in place along this coordinate is monitored. From these forces, a PMF can be calculated. Shown is C2 from PTEN bound to an anionic membrane [21]. (C) Complex PMF. PMFs calculated for binding of PH domains with, respectively, 1, 2, 3, 5 and 10 PIP₃ bound [120]. (D) Thermodynamic cycle as used in FEP for switching between PIP₃ and POPS for a PMP system. The difference between ΔG_{PIP3-POPS(bound)} and ΔG_{PIP3-POPS(free)} is the relative difference in PMP–lipid interaction for PIP₃ and POPS. Below are the values from preliminary analyses of Grp1 PH-binding FEP calculations. Note that these data were generated for this review, with a full manuscript to follow.