Yes, I think you are right--the somewhat counterintuitive case I was thinking of was, for example, when: Kd = 20nM [L] = 20uM [Po in crystal] = 20mM In this case, even though [L] = 20uM, since [L] is 1000 x Kd, the occupancy should be ~100%, and [PL] at equilibrium should be about 20mM, so in the crystal, the total [L] should be ~20mM. This explains, among other things, why bromophenol blue makes crystals bluer than the surrounding solution--the Kd is probably significantly lower than the BB concentration in the drop. Thanks for your clarifications! Jacob The question would remain, then, whether there is any utility in titrating ligands into crystals, and monitoring occupancies as a readout for binding. Although crystallization conditions are horribly non-physiological, perhaps there would be utility in the case where there are multiple known binding sites of various affinities, and other methods would have trouble resolving the binding events. One could start with: 1. totally saturated conditions, set occ=1 for all sites, refine B's, then 2. fix B's at this value, and refine the occ's in a subsequent series of dilutions. All of this is not totally theoretical--I am considering a set of experiments along these lines, where there really are multiple sites of varying affinity. ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: [log in to unmask] *******************************************