To further validate our findings for more realistic systems, we performed similar calculations and analyses on the Tyk2 emj42 ligand system. Table presents the average and standard deviation of dU dλ calculated from eight independent 10 ns simulations. The t-test p-values for the emj42 system, derived from comparisons of each timestep (1 fs, 2 fs, 2.5 fs, 3.3333 fs, and 4 fs) against the 0.5 fs reference (Table ), exhibit a similar trend. While the 1 fs and 2 fs timesteps generally maintain p-values above the significance threshold of 0.05, indicating statistical equivalence to the 0.5 fs results, the 4 fs timestep shows marked deviations with p-values well below 0.05. This indicates statistically significant differences in dU dλ The averages and standard deviations are calculated based on eight independent 10 ns NPT TI simulations conducted for each λ state. A total of five λ states (0, 0.25, 0.5, 0.75, and 1) and six timesteps (0.5, 1, 2, 2.5, 3.3333, and 4 fs) are used. Two thermodynamic states of the alchemical transformation of the Tyk2 ligand (ejm 42 to ejm 55) were studied: the aqueous state and the protein complex bound state.