In this study, the simulations of all wild-type and glycoform structures were performed using GROMACS 2020.4 at 310.15 K, which was in agreement with experimental temperature. Outputs in AMBER formats generated by GLYCAM glycoprotein builder were converted into GROMACS formats using ACPYPE to serve as the inputs of the simulations. Each structure was solvated in a dodecahedral box with 1.0 nm between the solute and the box edge. Sodium and chloride ions were added to neutralize the system and match the specified salinity of 0.15 M. The system was then energy minimized with the steepest descent algorithm until the maximum force was lower than 100.0 kJ/mol/nm. Subsequently, a 200 ps NVT equilibration followed by a 200 ps NPT equilibration was carried out, in which the Berendsen barostat and velocity rescaling were employed to maintain the reference temperature and pressure at 310.15K and 1 bar, respectively. Finally, an MD simulation was performed in an NPT ensemble, with the pressure maintained at 1 bar by the Parinello-Rahman barostat . The cut-off distances for van der Waals interactions and Coulomb interactions were both set as 0.9 nm, with the switching of the van der Waals potential between 0.85 and 0.9 nm and an analytical correction for long-range dispersion. The particle mesh Ewald algorithm was used with real-space switching of the potential between 0.89 nm and 0.9 nm. LINCS was used to constrain hydrogen bonds. All the simulations were extended up to 2000 ns, which we deemed necessary to capture insulin dynamics in which the major transitions between metastable states occurred on the time scale of 500-2000 ns, as interpreted from the time series of the pairwise RMSD calculations of the wild-type structures (see Supplemental Figure ). Our simulations captured the key important disordered elements investigated by Busto-Moner et al. , though at different percentages. This discrepancy is not surprising given the pH difference between the two studies, with the differences elaborated in Section 1 in the supporting information. All trajectories were stored every 250 ps, for a total of 8001 frames for analysis. All the input configurations and GROMACS mdp files are provided in the GitHub repository for this study.