This method determines the optimal equilibration time to be that which maximizes an uncorrelated sample size that can be obtained from an equilibrated gradient time series. After equilibration the 𝑑𝑉 𝑑𝜆 gradient time series was decorrelated (see Methods); then split in half chronologically and each half binned into seven equally spaced bins. The Jensen-Shannon (JS) distance, a measure of distance between two probability distributions, between these two histograms was calculated.