Fig. 5.

MD simulations validate the influence of hinge motion on the substates of the closed Met20 loop. (A) Simulation model of ecDHFR:NADP⁺:FOL highlighting the distance restraint applied in MD simulations between Asn23-$C_{\alpha }$ and Pro53-$C_{\alpha }$ (black dashed line) to model the effects of constricting (blue arrows, cold-like by analogy to the multi-temperature experiment) or expanding (red arrows, hot-like) the active site cleft. Kernel density estimates of the (B) hinge distance being restrained, (C) the Trp22-$\varphi$, and the (D) Met20-${\chi }_1$ dihedrals. The Trp22-$\varphi$ and Met20-${\chi }_1$ dihedrals, which report on the Met20 closed substates, show a monotonic response to the distance restraint. The kernel density estimates were produced from 100 independent simulations of 100-ns duration at each restraint distance.