Re: [ccp4bb]: substrate soaking into crystals

[<tsr@duke.edu>]

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On Wed, 12 Feb 2003, N.Manoj wrote:

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> Dear all,
>            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.
> Does anyone know of a case where the ammonium sulphate has been
> exchanged or replaced from the crystals by soaking etc and found
> competing substrates being bound after that?
> Thanks in advance
>
> manoj
>
>
>
manoj
	I've had a similar problem with my protein.  It is crystalized in
1.8 M ammonium sulfate.  On of the substrates is a disphosphate-coupled
lipid, and the sulfate anions compete for binding in the 'diphosphate
binding pocket' in the active site.  I have successfully transferred my
crystals from ammonium sulfate to sodium citrate through a steady crystal
soaking protocol, described (as best I can in email format) below

step 1       step 2        step 3                      step 7
1.8 M AmSO4  1.5 M AmSO4   1.2 M AmSO4 ... etc.,etc.   0 M AmSO4
0 M NaCit    0.15 M NaCit  0.3 M NaCit ... etc.,etc.   0.9 M NaCit


Hope this is of some help

Scott
tsr@duke.edu