  We describe a two-step approach for combining interactive molecular dynamics
in virtual reality (iMD-VR) with free energy (FE) calculation to explore the
dynamics of biological processes at the molecular level. We refer to this
combined approach as iMD-VR-FE. Stage one involves using a state-of-the-art
iMD-VR framework to generate a diverse range of protein-ligand unbinding
pathways, benefitting from the sophistication of human spatial and chemical
intuition. Stage two involves using the iMD-VR-sampled pathways as initial
guesses for defining a path-based reaction coordinate from which we can obtain
a corresponding free energy profile using FE methods. To investigate the
performance of the method, we apply iMD-VR-FE to investigate the unbinding of a
benzamidine ligand from a trypsin protein. The binding free energy calculated
using iMD-VR-FE is similar for each pathway, indicating internal consistency.
Moreover, the resulting free energy profiles can distinguish energetic
differences between pathways corresponding to various protein-ligand
conformations (e.g., helping to identify pathways that are more favourable) and
enable identification of metastable states along the pathways. The two-step
iMD-VR-FE approach offers an intuitive way for researchers to test hypotheses
for candidate pathways in biomolecular systems, quickly obtaining both
qualitative and quantitative insight.
