Fig. 5. Binding-free-energy measurements using BFEE2 software.

a, Free-energy difference for the double annihilation of heparin hexasaccharide in its bound state and in water from FEP calculations. λ is a coupling parameter that gradually switches on interactions between a fully coupled state (λ = 0) and a final, fully decoupled state (λ = 1). b, PMF obtained during the separation of hFGF1 and heparin hexasaccharide using the geometric route. We used the BFEE2 (ref. 27) package to estimate the absolute free energy of binding in silico for an alchemical or geometrical route with multiple subprocesses and geometric constraints. The alchemical and geometric methods’ binding-free-energy estimates were 0.55 ± 30.25 kcal mol⁻¹ and −19.04 ± 2.95 kcal mol⁻¹, respectively. See Supplementary Tables 2 and 3 for details of each geometric restriction and alchemical FEP calculations, respectively.

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