To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Kd's in Crystals
I had success using crystallography to measure the Ca2+ affinity (in the mM
range) for a Ca2+ dependent enzyme.
See:
Characterization and implications of Ca2+ binding to pectate lyase C.
Herron SR, Scavetta RD
Jacob,
In case if the hint that I sent yesterday was not clear, below is the solution
for the equation
Kd=[P][L]/[PL]
in terms of ligand occupancy:
O=[ PL]/[Po]= 1/(Kd/L+1)
You see, it does not depend on [Po]
Alex
On Jun 26, 2011, at 10:05 AM, aaleshin wrote:
The concentration of a
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
Hi,
We had a paper where we looked at Kd of arginine in the arginine
repressor-DNA complex (p. 248-249).
JMB,2010, *399*, pp.240-254.
Maia
Jacob Keller wrote:
Yes, I think you are right--the somewhat counterintuitive case I was
thinking of was, for example, when:
Kd = 20nM
[L] = 20uM
[Po
Jacob,
In the formula:
Kd=[P][L]/[PL]
[P] and [L] are concentrations of UNBOUND protein and ligand, and [PL] is that
in the complex.
Since the occupancy of the ligand in the crystal is
[ PL]/[Po]= 1/(Kd/L+1),
varying [L] around Kd like from 0.1Kd to 10Kd will make the titration of
occupancy.
Hi,
You may want to have a look at the two papers below.
Experimental determination of van der waals energies in a biological system.
Wear MA, Kan D, Rabu A, Walkinshaw MD. Angew Chem Int Ed Engl.
2007;46(34):6453-6.
The First Direct Determination of a Ligand Binding Constant in Protein
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
--
From: Jacob Keller j-kell...@fsm.northwestern.edu
Sent: Friday, June 24, 2011 6:58 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Kd's in Crystals
Dear Crystallographers,
what is the dogma with regard to affinities in crystals? For example,
if I soak three crystals in 1pM, 1nM
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
Dear Crystallographers,
what is the dogma with regard to affinities in crystals? For example,
if I soak three crystals in 1pM, 1nM, and 1uM compound X, and they all
show equivalent density, does that mean that the affinity is really
better than 1pM, or is the crystal of such a high local
Jacob Keller wrote:
Dear Crystallographers,
what is the dogma with regard to affinities in crystals? For example,
if I soak three crystals in 1pM, 1nM, and 1uM compound X, and they all
show equivalent density, does that mean that the affinity is really
better than 1pM, or is the crystal of such
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