Challenges Encountered Applying Equilibrium and Non-Equilibrium Binding Free Energy Calculations

13 April 2021, Version 2
This content is a preprint and has not undergone peer review at the time of posting.


Binding free energy calculations have become increasingly valuable to drive decision making in drug discovery projects.
However, among other issues, inadequate sampling can reduce accuracy, limiting the value of the technique.
In this paper we apply absolute binding free energy calculations to ligands binding to T4 lysozyme L99A and HSP90 using equilibrium and non-equilibrium approaches. We highlight sampling problems encountered in these systems, such as slow side chain rearrangements and slow changes of water placement upon ligand binding. These same types of challenges are likely to show up in other protein-ligand systems as well and we propose some strategies to diagnose and test for such problems in alchemical free energy calculations. We also explore similarities and differences in how the equilibrium and the non-equilibrium approaches handle these problems. Our results show the large amount of work still to be done to make free energy calculations robust and reliable and provide insight for future research in this area.


binding free energy
nonequilibrium free energy calculations
equilibrium free energy calculations
T4 lysozyme L99A

Supplementary materials



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