where x Box , y Box , and z Box are the side lengths of the simulation box, and x, y, z, ψ, θ, and ϕ are the Cartesian coordinates of the centre of mass and the Euler angles of the ligand in the frame of reference of the receptor. This is evaluated by taking the average intramolecular coordinates of the anchor points from a simulation of State 4. The receptor and ligand are then assumed to be rigid, allowing U r (x, y, z, ψ, θ, ϕ) to be calculated by translating and rotating the ligand anchor points with respect to the receptor anchor points and evaluating U r, Rigid (r 1 , . . . , r N ) = U r, Rigid (x, y, z, ψ, θ, ϕ) at each point, where "Rigid" shows that the anchor points have been fixed to their average intramolecular positions. This assumes that ∆G Preorg. = 0 (Equation ), and produces a bias towards more negative free energies of binding, because ∆G Preorg. ≥ 0. The error will be substantial when the average intramolecular positions of the anchor points for the decoupled complex are very different to those in the free ligand and receptor, or the restraints substantially restrict the conformational freedom of the receptor or ligand, thus enforcing substantial "preorganisation".