We investigated the outliers in this system to try and identify potential sampling problems. All transformations between the amide and the biaryl series had calculated ∆∆G values that were too unfavorable (SI Figure ). Extending the length of the simulation from 10 ns per λ window to 20 ns improved the results slightly (RM SE ∆∆G = 2.23 kcal mol -1 vs. RM SE ∆∆G = 1.83 kcal mol -1 ), suggesting that simulations had not reached convergence yet. Preliminary free energy calculations initiated from different conformations of the 10s loop (PDB ID: 4FS4 vs. 6OD6) showed that results substantially depend on the starting structure since the conformational change was not sampled adequately.