Figure 2. Schematic illustration of three key enhanced sampling methods.

In all cases, the black line represents a free energy landscape projected onto a single CV, for simplicity. (a) Replica exchange MD, in which multiple independent replicas are run under different conditions, such as at increasingly high temperatures (red to yellow lines), which smooth the free energy landscape; (b) Umbrella sampling, where the blue harmonic potentials represent the ‘umbrellas’ that restraint conformational sampling along the CV; (c) metadynamics, where the potential energy surface is smoothed along one or more CVs by adding Gaussian functions (blue) to regions of the conformational space that have already been visited until ultimately (cyan) the entire surface is filled; (d) well-tempered metadynamics, where the rate and size of the Gaussian functions (blue) are reduced as sampling progresses, resulting in a smooth free energy surface (or a pre-specified distribution function, cyan) and avoiding over-filling.