The crystal structure of Anc4 + Loop II integrated with all six substitutions (p12K, n13H, s58T, n193G, y194F, and d197R) revealed the interactions of these residues with loop II. Substitutions were introduced gradually due to the convenience experimentally as mutations A (cyan), p12K and n13H; mutation B (slate), s58T mutations C (magenta), n193G, y194F, and d197R. Small and large characters indicate the Anc4 and Anc5 states, respectively. Side chains and Cα atoms of the introduced residues are shown as stick and spheres, respectively.
