Another way to determine the free energy difference between binding modes is to use umbrella sampling. Similar to AFE, umbrella sampling constructs a series of intermediate states between binding modes along a physical degree of freedom. For example, for sampling the energy change associated with rotating a rotatable bond, the intermediate states are described by different degrees of rotations of the rotatable bond. The system is simulated at the intermediate states using restraints, and then a free energy profile is constructed based on all sampled conformations. However, sampling along a single degree of freedom can result in inadequate sampling of all relevant confor-mations in complex three-dimensional proteinligand systems. Particularly, orthogonal degrees of freedom can be especially difficult to adequately sample. Both AFE and umbrella sampling simulations require prior knowledge of what the different binding modes are.