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
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