Re: [ccp4bb]: substrate soaking into crystals

[Gregory Verdon <g.verdon@chem.rug.nl>]

Hi,

Yes, the sulfate is probably competing with ATP for the binding to the

phoshate groups binding sites. Actually, i would guess that the map shows clearly a sulfate

ion bound in the nucleotide-binding site. You may try to do soaking experiments with

a really high concentration of ATP (about 10, 20, 50 mM ...). May be you can also

decrease the sulfate concentration in your soaking solution (to 0.1 M or 0.05 M).

The other alternative is to replace the ammonium sulfate by something else in the a

soaking solution (NH4Cl or NaCl).

If you don't see the sulfate ion bound in the nucleotide-binding site, then the problem

is different: the nucleotide-binding-site is not accessible or the affinity for ATP is very low !

May be you have need to add Mg2+ (if your protein use Mg2+ to hydrolyse ATP, in this case of course

you will get ATP-hydrolysis, ...). Then you could try with AMPPNP-Mg2+ or AMPPCP-Mg2+ !!!

Have you tried to soak with ADP-Mg2+ ?

Good luck !

                        Greg
 
  ________________________________________________________________
                                                                
  Gregory Verdon                                                                                                                                   
  Laboratory of Biophysical Chemistry                                     
  Department of Chemistry                                                  
  University of Groningen                                                  
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