Relative binding free-energy perturbation (RBFE) compares the binding affinities of related ligands by alchemically transforming one into another in both the protein complex and solvent. The difference between these transformations gives the relative binding free energy, ∆∆G.
Settings
Inputs: a prepared protein and related ligands in aligned bound poses, with substantial structural overlap and the same net charge. Experimental poses are preferred; docking or co-folding can introduce pose errors. Unrelated ligands may need endpoint methods or ABFE.
Aligned poses: if starting from analogue SMILES, run analogue docking first and include the reference ligand's SMILES explicitly. Inspect overlaid poses and MCS RMSD before building the graph.
Graph mode: "Greedy" connects similar ligands with redundant paths. "Star Map" connects every ligand to the selected "Hub compound," using fewer edges but providing no cycle checks.
Scoring method: "Jaccard" uses atom-mapping similarity; "Dummy atoms" uses the number of unmatched atoms. "Best" (default) tries both and selects the graph with fewer dummy atoms.
K min cut: target graph redundancy, default 3, for greedy graphs. Higher values generally add edges and cost. "Generate intermediate ligands?" can split large transformations into smaller ones.
Cofactors: list fixed small molecules in the protein structure that need parameterization, with residue identifiers and SMILES. Known ions and waters do not need SMILES.
Forcefield / Charge method: choose ligand parameters and partial charges. The web default uses OpenFF Sage 2.0.0 with NAGL; NAGL is faster than AM1-BCC for large ligands. The web form requires NAGL for Mango.
Equilibration steps / Num frames: defaults are 200,000 equilibration steps (0.5 ns) and 2,000 production frames (2 ns) per window, at 400 steps per frame and a 2.5 fs timestep.
Initial lambda windows / Minimum overlap / Target overlap: defaults are 48 maximum windows and 0.667 for both overlap targets. Adaptive scheduling uses fewer windows for easier transformations.
Local MD steps: default 390 of each 400-step frame; 0 disables local resampling. "REST max temperature scale" defaults to 1 (disabled); higher values enable solute tempering.
Resubmit the completed RBFE graph as FEP and inspect or edit its edges. "Recommended (Default)" uses the settings above; "Fast" shortens sampling and lowers overlap targets. "Rigorous" disables local resampling and uses AM1-BCC charges.
Notes
Run the "System compatibility check" to catch missing atoms, nonstandard residues, or incompatible protein naming. Membrane embedding and covalent bond formation are unsupported; compare only the noncovalent contribution of covalent inhibitors. Compare different ligand charge states in separate groups.
Results include per-edge ∆∆G and uncertainties, per-ligand relative affinities in kcal/mol, cycle closure, overlap, convergence, and replica mixing. Saved trajectories cover the complex and solvent endpoints. Inspect these diagnostics before interpreting affinities; uncertainty estimates do not capture all pose, force-field, or sampling errors.
For comparison with experimental absolute affinities, add a constant to align dataset means before calculating MAE or RMSE. The walkthrough uses a Rock2 system from Uni-FEP. Rowan does not propose analogues automatically; CReM is one possible source.
Submission video
Benchmarks and validation
Compare binding-affinity predictions across JACS, Merck, Waterset, and additional systems. Select a benchmark to inspect the individual compounds: free-energy-perturbation benchmarks.