Dear Gourab,
Sorry for the delayed response, I missed your message earlier.
Can you grow the protein crystals easily (do you have lots of them)?
Is your ligand soluble in PEG, MPD, or Solutol?
Often, you can exchange the crystal solution without destroying the
crystal. But, you have to match the water activity of the original
growth solution with the new solution. This way you could get your
ligand into one of these non-salt solutions and then move a crystal from
your grow solution into the new solution with your ligand. This would
expose your crystal to the ligand.
Obviously, you will probably burn through several crystals to see what
PEG, MPD, of Solutol concentration your crystals will tolerate. To some
degree, this is no different than hunting for the best cryo-condition.
Thus, I would see which solution your ligand prefers first, so you can
limit your crystal trials.
I worked on an enzyme that required calcium for activity, but we could
only grow the crystals in ammonium sulfate. I was able to exchange the
crystal growth solution for a PEG solution. The protein has some
affinity for the sulfate ions, so the exchange needed to be done over a
~4 hour period of time. If I went faster, the sulfate would remain in
the crystal channels and would precipitate when exposed to calcium. It
was tedious working this out, but it was essential for everything else
that came later.
Steven Herron [sherron_...@yahoo.com]
On 4/23/2021 11:40 PM, Gourab Basu Choudhury wrote:
I tried sokaing ,high concentration of DMSO is effecting crystal, so I
tried other contidtion where 10%DMSO is there. Got 1.9 A data with
protein co crystalization with 10mM ligand. But ligand occupancy is
not visble.
I got the KD value from ITC, with reverse titration. With 10uM ligand
in cell and 150uM protein in syringe. .
Can anybody suggest some views.please feel free to share your experiences.
On Sat, Apr 24, 2021, 10:49 AM Peat, Tom (Manufacturing, Parkville)
<tom.p...@csiro.au> wrote:
Hello Gourab,
DMSO is not the only possible solvent- there are others to try.
I don't want to sound negative, but 40 micromolar is not a very
tight binding compound. It would also depend on how that binding
constant was measured- how did someone get enough in solution to
measure that?
Best of luck, tom
Tom Peat, PhD
Proteins Group
Biomedical Program, CSIRO
343 Royal Parade
Parkville, VIC, 3052
+613 9662 7304
+614 57 539 419
tom.p...@csiro.au
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*From:* CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK
<mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of Gourab Basu Choudhury
<goura...@csiriicb.res.in <mailto:goura...@csiriicb.res.in>>
*Sent:* Saturday, April 24, 2021 12:46 PM
*To:* CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>
<CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>>
*Subject:* [ccp4bb] Co crystalization with less soluble ligand.
Hello everyone,
I am finding it difficult for getting a ligand density inside the
protein as the ligand is very much insoluble. It's only soluble in
100% DMSO. I tried for co crystalization. Kd value of the ligand
is near 40um. Any suggestion what to do?
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