Computational methods were used to determine the three-dimensional structure of the Agitoxin (AgTx2) -Shaker complex. A large number of models of the complex were generated using high temperature molecular dynamics, accounting for side chain flexibility with distance restraints deduced from thermodynamic analysis of double mutant cycles. The quality and validity of the resulting complexes was assessed by examining the stability of the binding modes during molecular dynamics simulations with explicit water molecules and by calculating the binding free energies of mutant proteins using a continuum solvent representation and comparing with experimental data. In all the models it was found that the side chain of Lys27 of the toxin binds deep into the pore, to the S1 cation binding sites.