Dear Afshan,
As Fred suggested, you may try to get rid of the ammonium phosphate by
transferring it to a PEG solution. However, it may be that you get the nice
crystals because your protein is stabilized by the phosphate in the active
site. Crystal packing may prevent the phosphate from leaving the active site,
or the crystals may crack when the the phosphate leaves the active site. When
transferring, I would transfer the crystals to a solution containing your more
potent inhibitor, so the phosphates may be exchanged by the inhibitor.
I do not know whether your 3.5 Å crystals were grown in the presence of the
inhibitor, but with a 1.75 Å native structure, you maps should show a bound
inhibitor at 3.5 Å, so this could already solve the question of the binding
mode.
Finally, you did not give details of your cocrystallization experiments. Did
you just try the native crystallization conditions in the presence of the
inhibitor? Crystallization conditions can be radically different in the
presence of an inhibitor and when the first attempts were unsuccesful, I would
do a full de novo crystallization screen (Hampton, PEG screen, whatever you
normally use) in the presence of the inhibitor.
Beste regards,
Herman Schreuder
________________________________
From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of
Afshan Begum
Sent: Friday, May 27, 2011 2:24 PM
To: [email protected]
Subject: [ccp4bb] need proper suggestion
Dear All,
I have a severe prob lam to performed my ligand binding study with
corresponding protein. I have taking the native diffraction data at 1.75 A and
after that i have performed soaking as well co-crystallization experiment with
my inhibitors.
Problem is that at the active site phosphate ion always bind instead of
inhibitors. I have used 1.6 M ammonium phosphate conc at the crystallization
recipe which is a very weak inhibitor of my protein whereas the ligand is
already clinically applicable but due to the very high conc. of phosphate i
have not achieved my target. If some one can suggest me what else i can replace
with ammonium phosphate or any other suggestions would be appreciated.
I have tried to grown crystals some other condition but the crystal was not
diffracted beyond 3.5 A.
Best Regards
AFSHAN
