To reduce sampling bias of our MD simulations and ensure that our analysis results were not dependent on the initial configuration of insulin, in our study, we built each of the 12 glycoforms on 5 wild-type (WT) structures resolved by different groups using different methods, whose PDB IDs were 4EYD, 4EY9, 4EY1, 3I3Z, and 2MVC, respectively (Figure ). These initial models were chosen based on whether they were resolved in a complex or as a monomer. For example, 4EYD, 4EY9, and 4EY1 were all crystallized in complex with Zn 2+ as a hexamer composed of a dimer in 3-fold symmetry . They represented high-resolution human insulin structures from pharmaceutical formulations. 3I3Z and 2MVC, on the other hand, were representative of monomeric insulin structures. 3I3Z was resolved by X-ray crystallography under low gravity conditions so that the asymmetric unit of insulin was a monomer . 2MVC was resolved by NMR spectroscopy under acidic conditions, which is known to favor the monomeric form of insulin .