In the SEEKR2 algorithm, the phase space of the receptor-ligand complex is partitioned into two distinct regions based on the distance between the ligand and the binding site of the receptor, i.e., the MD region and the Brownian dynamics (BD) region (Figure ). SEEKR2 employs the OpenMM engine to run the MD simulations 106,107 and the Browndye2 engine to run the BD simulations . The MD region is in close proximity to the bind- ing site, where solvent effects and molecular flexibility predominantly mediate the receptor-ligand interactions. Consequently, fully atomistic MD simulations are required in this region to represent the molecular interactions accurately. A Voronoi tessellation approach partitions a given region into multiple smaller regions based on the proximity to a set of anchor points . In a Voronoi diagram, the boundaries of the cells corresponding to an anchor point are equidistant to adjacent anchor points, and the vicinity corresponding to the given anchor point is closer to that point than any other input point (Figure ). Figure illustrates a Voronoi diagram for a simple Muller potential system transitioning between states A and B using a CV that follows a likely reaction pathway between A and B. The CV is segmented into anchor points. Milestones are defined as surfaces that are equidistant between two adjacent anchor points. The spaces between milestones constitute the Voronoi cells. A Voronoi cell around a particular anchor point is the region in configuration space where every point within this cell is closer to that anchor point than any other. With the SEEKR approach on a biologically relevant receptor-ligand system, the MD region is partitioned into "Voronoi cells" based on the distance between the center of mass (COM) of the ligand and the COM of the alpha-carbon (α-C) atoms of the binding site residues of the receptor. Each Voronoi cell contains a copy of the system where the ligand is at varying distances from the binding site of the receptor, where independent MD simulations are carried out. To generate starting structures of the receptor-ligand complex in each Voronoi cell, steered molecular dynamics (SMD) simulations are propagated that facilitate the controlled movement of the ligand away from its binding site and into the solvent. This process involves gradually pulling the ligand out of the binding pocket with a moving harmonic restraint while maintaining the system in equilibrium and adding no significant stress. Each time the ligand crosses a milestone, a snapshot of the receptor-ligand complex is saved as a starting structure for that particular Voronoi cell for running SEEKR2 simulations. MD simulations with the Markovian milestoning scheme are then employed concurrently within individual Voronoi cells until convergence is reached . Reflective boundary conditions are implemented to confine the molecular trajectories within each cell. The mean free passage time (MFPT) is then calculated from the transition matrix, ∧ Q obtained from the MMVT-SEEKR2 simulations. Section 2.2.2 provides a comprehensive set of equations employed in the calculation of mean free passage time (MFPT) and the binding free energy profile (∆G i ) in the context of SEEKR2 simulations.