The complex is then subjected to backrub Monte Carlo steps for 50,000 steps at a temperature of 1.2 kT, producing 50 different models. The backrub move implemented in Rosetta samples local side chains and backbone conformational changes within 8 Å 2 of the mutated position. Each model is then packed to optimize side chain conformations using the Rosetta packer move, and minimized. In parallel, each model is mutated at the selected position using a pre-determined substitution. As well as the wild-type model, the mutated models are then packed and the energy of the system minimized. Finally, all the wild-type and mutant models are scored in complex, as well as the complex partners being scored individually. The scores of the complex partners alone are obtained simply by moving the complex partners away from each other. No further minimization or side chain optimization is performed on the unbound complex before scoring. The interface ∆∆G is calculated using the following equation, as the arithmetic mean over the 50 models produced: