The bacterial phosphotransferase system couples phosphoryl transfer to sugar transport across the cell membrane. The first protein in the pathway, Enzyme I (EI), undergoes two large rigid body domain reorientations between an autophosphorylation-competent closed state and an open state that allows subsequent phosphoryl transfer to its downstream protein partner. Simultaneous use of solution X-ray scattering and NMR dipolar coupling data to guide simulated annealing refinement reveals the existence of a dynamic equilibrium between closed and partially closed conformations in a complex of a mutant of EI with phosphoenolpyruvate. The partially closed conformation represents an intermediate in the open-to-closed transition.