Mutations were then introduced into each of these structures using pmx . To reduce the likelihood of clashes between the 'new' sidechain and the remainder of the protein (i.e. in simulations with λ~1) we then applied a short Alchembed procedure to each structure -this involved a 1,000 step simulation where λ was increased from 0 to 1 using a soft-core van der Waals potential. This created a pool of presumed independent mutated structures that could be used to seed alchemical thermodynamic integration simulations.