Timesteps greater than 2 fs might lead to inaccurate dU dλ data a The averages and standard deviations are calculated based on eight independent 10 ns NPT TI simulations conducted for each λ state and softcore condition. A total of five λ states, i.e., 0, 0.25, 0.5, 0.75, 1, and six-time steps, i.e., 0.5, 1, 2, 2.0, 2.5, 3.3333, and 4 fs, is performed. Three cases of alchemical transformation of the cAMP system were studied: without a softcore (No SC), with only one oxygen atom transformed into a sulfur atom (1 atom SC), and with all atoms undergoing softcore transformation (all SC). The averages and standard deviations are calculated based on eight independent 10 ns NPT TI simulations conducted for each λ state and softcore condition. A total of five λ states, i.e., 0, 0.25, 0.5, 0.75, 1, and six-time steps, i.e., 0.5, 1, 2, 2.0, 2.5, 3.3333, and 4 fs, is performed. Two cases of alchemical transformation of the ethane to ethanol transformation were studied: from -CH 3 to -CH 2 OH (4/5 atoms SC) and with all atoms undergoing softcore transformation (all SC).