Sensitivity of Binding Free Energy Calculations to Initial Protein Crystal Structure

Binding free energy calculations using alchemical free energy (AFE) methods are widely considered to be the most rigorous tool in the computational drug discovery arsenal. Despite this, the calculations suffer from accuracy, precision, and reproducibility issues. In this publication, we perform a high-throughput study of more than a thousand AFE calculations, utilizing over 220 mu s of total sampling time, on three different protein systems to investigate the impact of the initial crystal structure on the resulting binding free energy values. We also consider the influence of equilibration time and discover that the initial crystal structure can have a significant effect on free energy values obtained at short timescales that can manifest itself as a free energy difference of more than 1 kcal/mol. At longer timescales, these differences are largely overtaken by important rare events, such as torsional ligand motions, typically resulting in a much higher uncertainty in the obtained values. This work emphasizes the importance of rare event sampling and long-timescale dynamics in free energy calculations even for routinely performed alchemical perturbations. We conclude that an optimal protocol should not only concentrate computational resources on achieving convergence in the alchemical coupling parameter (lambda) space but also on longer simulations and multiple repeats.

Automated summary

This study investigated the impact of initial crystal structures on binding free energy values in alchemical free energy calculations, finding that the initial structure can significantly affect values obtained at short timescales. Rare events, such as torsional ligand motions, became important factors at longer timescales, leading to higher uncertainty in the obtained values. Optimal protocols should focus on achieving convergence in the alchemical coupling parameter space, as well as longer simulations and multiple repeats.