Determination of thermal intermediate state ensemble of box 5 with restrained molecular dynamics simulations

► The “invisible” intermediate state was predicted with ensemble-averaged simulations. ► Distance and angle restraints from experiments were imposed on the simulations. ► The structural characters of intermediate state ensemble were depicted in detail. ► The ensemble was high heterogeneity and L-shaped topology was vanished. ► Helix 3 was more native-like and the hydrophobic core was unformed completely.