Simulated structures from 4EYD, 4EY1, and 3I3Z had exactly the same protonation state for each residue. 4EY9 and 2MVC, on the other hand, were found to have different histidines protonated compared to the ones in the other three models (see Supplemental Table ). We chose to include this ensemble of protonation sites, as they were essentially attributable to the orientations of the histidine residues and their surroundings. In addition, these two histidine residues were far away from the residues involved in the hypotheses of our analysis methods (see Supplemental Figure ), making them less likely to have noticeable influences on the predictors we developed. We started from these parameterized wild-type insulin confor-mations with reasonable protonation states and used GLYCAM glycoprotein builder , which used the GLYCAM06j-1 force field, to build various glycoform structures by attaching different saccharide moieties to the corresponding glycosylation sites.