Here, we demonstrate a method to estimate the uncertainty (confidence intervals and standard errors) of free energy differences calculated by molecular simulations. The widths of confidence intervals and standard errors can be calculated solely from temperature and the number of transitions between states. Uncertainty (95% confidence interval) lower than ±1 kcal/mol can be achieved by a simulation with four forward and four reverse transitions. For a two-state Markovian system, the confidence interval is exact, regardless the number of transitions.

Department of Biochemistry and Microbiology University of Chemistry and Technology 166 28 Prague Czech Republic

Faculty of Mathematics and Physics Charles University 186 75 Prague Czech Republic

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Analysis of metadynamics simulations by metadynminer.py