The concentration of a protein in a crystal [Po] and the volume of a crystal V are needed only to calculate the total amount of a ligand [Lo] required for soaking. [Lo] > [Po]*V
The occupancy of the active sites in a crystal will depend only on the ligand concentration in solution and Kd. It does not depend on protein concentration in the crystal. Indeed: Kd=[P][L] / [PL] (chemical equilibrium equation) Assuming total concentration of the protein = Po, Kd= 1mM and S= 1 mM, the active site occupancy will be: 1= P/Po-P; P/Po=1/2 So the concentration of the ligand in solution should be >>Kd to get the full occupancy. Alex On Jun 25, 2011, at 9:19 PM, Jacob Keller wrote: > Upon some reflection, I think one can say this: first, let's say the > protein in question is 30kD, with a solvent content of 50%, and we > know that solid protein density is ~1200mg/mL. Therefore, the protein > concentration in the crystal would be ~20mM. Because Kd's assume > infinitesimal ligand concentration, I think that neglecting ligand > depletion effects mentioned by Edward Berry, say by having a huge > reservoir or transferring the crystal to an appropriate soaking > environment, that all ligands which bind with a better than ~20mM Kd > should be bound in that crystal, even at extremely low ligand > concentrations, so changing [ligand] from 1pM to 10mM should not > change occupancy much, again assuming equilibrium and neglecting > ligand depletion. > > JPK
