An alternative to docking which is more rigorous, but computationally expensive, is to apply free energy calculations based on molecular simulations to predict populations of possible stable binding modes. For example, the "confine and release" approach allows multiple binding modes to be treated separately by distinct free energy calculations, and then subsequently combining the individual binding free energies to yield a total binding free energy (Figure ). Specifically, the overall binding free energy of a ligand to a protein can be decomposed into a particular type of average over the effective binding free energies of different metastable binding modes. As long as these metastable binding modes are defined a way that they cover the full bound state including all the relevant binding modes, and they do not overlap in phase space, this approach is rigorous. However, the number of required binding free energy calculations scales linearly with the number of binding modes for this already computationally demanding approach, making it unappealing to consider multiple candidate binding modes separately in this manner. This approach been exemplified recently in binding free energy studies on bromodomains; multiple binding modes were considered separately. The crys-tallographic binding mode was always found to have a higher affinity than other metastable binding modes. A similar approach has also been used with empirical free energy methods