For those models resolved in the dimeric form, including 4EYD, 4EY9, and 4EY1, we extracted the insulin monomer from the dimer conformation. For 3I3Z, which was resolved as a monomer with the last residue on the B-chain mutated into an alanine, we used PyMOL to mutate the residue back to a threonine to match the standard sequence of human insulin. Lastly, for the NMR-resolved 2MVC, we simply took the first model from the PDB file of the resolved ensemble. Notably, even if monomers extracted from a dimer conformation could potentially suffer from biases from experimental conditions that favor dimer resolution, as later shown, these biases did not have noticeable influences on the consistency of our methods. After extracting the monomer structure from each of the initial models, we used the H++ server (version 3.2) , which also parameterized the protein with the AMBER ff14SB force field, to assign reasonable protonation states corresponding to the pH values adopted in experiments. Specifically, in the work by , the experiments of 𝛼-chymotrypsin digestion (for estimating the 𝛼-chymotrypsin half-life, hence the proteolytic stability) and analytical ultracentrifugation (for estimating the monomer/dimer population, hence the dimerization propensity) were conducted at pH 8.0 and pH 7.4, respectively. These two pH values respectively correspond to the typical pH values in the pancreatic tract (pH 7.5 to pH 8.0 ) and small intestine (pH 6 to pH 7.4 ) of human body. Around these pH values, the total charges of insulin were predicted to be -1 or -2, depending on the pK 1∕2 value of the two histidine residues (HisB5 and HisB10) of insulin. We then adjusted the external pH value in H++ of each wild-type structure to give total charges of -1. Notably, this value of total charge was preferred to -2 because it collectively corresponded to pH values ranging from 6.6 to 7.8 (see Supplemental Table ), which overlapped both the pH ranges in the small intestine and the pancreatic tract. If a total charge of -2 were adopted, the corresponding pH ranges would have been higher and not able to overlap the typical pH range of the small intestine.