(2) The highly conserved second-shell amino acid side chain anion of E97, which forms an ion pair to the cationic K12 side chain. The E97A substitution examines the modelling of replacement of a residue on the second-sphere of side chains at the enzyme active site. (3) The side chain of P166, which clashes with the carbonyl oxygen of G209 in loop 7 during the ligand driven movement of the loop from its unliganded open to its ligand-bound closed conformation. This steric clash results in movement of the E165 carboxylate from an initial "swung-out" position to a "swung-in" orientation that points into the TIM active site, where it is poised to deprotonate the enzyme-bound substrate. The P166A substitution eliminates this steric clash so that the E165 side chain remains in the swung-out position in the closed form of TIM. This amino acid substitution tests the precision of EVB simulations in modelling the effect of an amino acid perturbation that results in a defined change in protein structure.