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 μs 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 (λ) space but also on longer simulations
and multiple repeats.