Re: [ccp4bb]: summary of sulphate binding at ATP binding sites

[Anastassis Perrakis <perrakis@nki.nl>]

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>>            I am trying to get cocrystals with ATP bound in the active
>> site.  But the electron density maps show no evidence of ATP binding. 
>> I
>> have ~200mM ammonium sulphate, PEG and 4mM ATP in the crystallization
>> conditions and most probably ammonium sulphate competes with the ATP,
>> preventing its binding.

Motivated by the load of answers, a few more questions /attempts for 
answers.

I can understand that in the absence of ATP/analogue AS will bind there.
I also understand that in a soaking experiment it might be hard to 
replace AS,
due to crystal packing and difficulties for the ATP to go where it 
should ...

BUT, if it's hard to replace AS by ATP (in a co-crystallization 
experiment,
as maybe I miss-understood,  on second reading maybe that seems
like a soaking experiment) isn't there something really more fishy 
going on ?
  I would expect affinity for ATP to be >>> than AS.
And, thats  4mM ATP against 200mM AS, not against 2-3 M AS
Also, it seems like I was the only one pointing the lack of Mg ions in 
the (reported) conditions.
Isn't this the thing to try first ? (unless they are there and not 
reported of course...)

In brief, I am trying to suggest that before trying all the nice 
protocols for transfer that
people have worked out during the years, i would:
0. make sure Mg is around ...
1. try co-crystallisation with ATP, NOT soaking
2. try ADP/AMPpNP/ATP-gamma-S, etc

THEN, I would try to get new crystallization conditions, in contrast to 
the conclusion that was drawn
by manoj, following the discussion.

	Tassos