Abstract
Here we introduce a high-throughput, formally exact method for absolute binding-free-energy calculations that enhances computational efficiency and accuracy. At the core of this method is a thermodynamic cycle that minimizes protein ligand relative motion, thereby reducing system perturbations and driving a fourfold gain in efficiency over the traditional double-decoupling method. By combining this strategy with double-wide sampling and hydrogen-mass repartitioning algorithms, the efficiency is further boosted to eightfold. The presented method is applied to 45 diverse protein–ligand complexes. For 34 complexes with validated force-field accuracy, our method achieves an average unsigned error of less than 1 kcal mol−1 and a hysteresis below 0.5 kcal mol−1, showcasing exceptional reliability. Moreover, it efficiently manages flexible peptide ligands through a potential-of-mean-force calculation, adding less than 5% extra simulation time. For 11 challenging cases, the presented method also shows an improvement compared with previously published results. Put together, this method has potential for advancing research in physical, biological and medicinal chemistry.
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Data availability
Source data for Figs. 1c,d and 2a–d,f and Extended Data Fig. 1c–e are provided with this paper. Source data for Fig. 2e are provided in Supplementary Table 3. The initial structures of the molecular assemblies are available from the Protein Data Bank (www.rcsb.org), with corresponding PDB IDs provided in Supplementary Table 1. The initial and final structures as well as the topology files for equilibrations are provided in Supplementary Data 1–3. A step-by-step tutorial for reproducing the free-energy calculations using BFEE3 and NAMD is included in Supplementary Section 4.
Code availability
Simulation input files were generated using BFEE3 and are available via GitHub at https://github.com/fhh2626/BFEE2 (ref. 41), and all simulations were performed using NAMD 3.0 (https://www.ks.uiuc.edu/Research/namd/, ref. 33). Both BFEE3 and NAMD are open-source tools. BFEE3 also facilitates all necessary postprocessing.
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Acknowledgements
H.F. acknowledges the National Natural Science Foundation of China (grant nos. 22473062 and 22293030), the National Key R&D Program of China (grant no. 2022YFA1305200) and the Natural Science Foundation of Tianjin (grant no. 23JCQNJC01420). W.C. acknowledges the National Natural Science Foundation of China (grant no. 22373051). X.S. acknowledges the National Natural Science Foundation of China (grant no. 22374082). C.C. acknowledges the European Research Council (project no. 101097272 ‘MilliInMicro’) for its support.
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H.B. and H.F. designed research. H.B., X.S., C.C., W.C. and H.F. performed research and analyzed data. H.B., C.C. and H.F. wrote the paper.
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Nature Computational Science thanks Yun Luo, Benjamin Ries and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Kaitlin McCardle, in collaboration with the Nature Computational Science team. Peer reviewer reports are available.
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Extended data
Extended Data Fig. 1 Additional step in binding free-energy calculations for flexible ligands and the convergence of alchemical transformation.
(A) Crystal structure of the Abl-SH3:p41 complex. (B) Comparison of bound and unbound conformations of p41. (C) PMF of free p41 using RMSD as the CV. (D, E) Forward-backward overlap of free-energy calculations for (D) 4HBV and (E) 1BBZ.
Supplementary information
Supplementary Information
Supplementary Figs. 1–20, Sections 1–5 and Tables 1–7.
Supplementary Data 1
Initial and final structures, as well as the topology files for equilibrations (part 1).
Supplementary Data 2
Initial and final structures, as well as the topology files for equilibrations (part 2).
Supplementary Data 3
Initial and final structures, as well as the topology files for equilibrations (part 3).
Source data
Source Data Fig. 1
Statistical source data (txt) for Fig. 1.
Source Data Fig. 2
Statistical source data (txt) for Fig. 2.
Source Data Extended Data Fig. 1
Statistical source data (txt) for Extended Fig. 1.
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Bian, H., Shao, X., Chipot, C. et al. A formally exact method for high-throughput absolute binding-free-energy calculations. Nat Comput Sci (2025). https://doi.org/10.1038/s43588-025-00821-w
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DOI: https://doi.org/10.1038/s43588-025-00821-w
