Overall, the predictions made out of the glycan-involved hydrogen bonds analysis are relatively consistent with the experimental results. The only exception is GF 6, which was experimentally found to be less proteolytically stable than the wild type but had a hydrogen bond, ThrB27(N)-GalNAc (O3) that involved a P2' site ThrB27. However, this hydrogen bond was only present in the MD trajectories of 4EYD-, 4EY1-, and 2MVC-based GF 6 (see Supplemental Table ), indicating that this hydrogen bond might not be stable enough to consistently disturb the aforementioned hydrogen bonding network. Importantly, the fact that 4EYD-, 4EY1-, and 2MVC-based GF 6 all possess the same hydrogen bond is one of many examples showing the large error bars should be irrelevant to the initial modeling bias. Specifically, if there is a bias from the differences in the resolution methods or the histidine protonation states, glycoforms resolved by the same method (e.g. 4EYD-, 4EY9-, and 4EY1-based glycoforms) or having the same histidine protonation states (e.g. 4EYD-, 4EY1-and 3I3Z-based glycoforms) should exhibit similar hydrogen bond distributions at the examined site. However, this trend is absent from our results, suggesting that the metric is independent of the starting wild-type models. Finally, we can conclude that long-lasting, stable glycan-involved hydrogen bonds, especially the ones that involve PheB24 and ThrB27, appear critical in enhancing the proteolytic stability of the structure. This conclusion also underpins the findings in the work by Guan et al. that ThrB27 and ThrB30 were identified as the best two glycosylation sites, as the glycan attached to these sites are structurally closer to the important residues (ThrB27 and PheB24) found in this study. respectively indicate that the corresponding glycoform was experimentally found to have higher, lower and comparable proteolytic stability as compared to the wild type. Note that in the name of each hydrogen bond, the 1-based index of the glycan is shown in the bracket following the name of the glycan. The atom type is shown in the parenthesis right after the residue name. (See Supplemental Table for more details of the atom types shown in the figure .) For example, ThrB27(N)-Man (O6) means the hydrogen bond formed between the amide N atom of ThrB27 as the donor and one of the oxygen atoms of the second mannose as the acceptor. Texts for hydrogen bonds that involve any of the P1-P3 or the P2' residues are colored in blue, which in our case only include PheB24 and ThrB27.