The absolute free energy of a system can be determined if we know its partition function (Q), a function that connects microscopic properties of the system with macroscopic thermodynamic quantities. Unfortunately, it is very hard to calculate the absolute free energy of real systems because we don't know their partition functions. Free energy calculations allow us to bypass this problem, but require at least two states: a reference state whose free energy can be analytically or numerically found, and a final state of interest . We chose to calculate the free energy difference using alchemical free energy calculations, a method in which we simulate a series of nonphysical intermediates between the end states .