In contrast to the positive cooperativity discussed above, entropy can also drive negative cooperativity. In the homotetramer transthyretin (TTR), the affinity for small molecules to the second site is weaker. However, the structure of each subunit, including the binding sites, looks structurally similar, posing the question of why there is a difference in affinity. CryoEM revealed an inherent asymmetry in the ensemble of conformations of each dimer and that the first binding event does not lead to ordering the second binding site. On the contrary, the subsequent binding event requires more extensive structural rearrangements, including loop ordering, which comes with at a higher entropic cost . Entropically driven negative cooperativity is also observed with NMR order parameter measurements in the bacterial transcription factor CzrA, which has a Zinc binding site more than 15 angstroms away from a DNA-binding site. Without Zinc, DNA binding increases side chain flexibility throughout the core of CrzA, increasing DNA binding affinity through favorable entropy. However, upon binding Zinc, side chain flexibility strongly decreases, and thus conformational entropy, eliminating this favorable entropy and thus reducing DNA binding affinity (Figure ) .