Dear All,
thanks for your replies. I tend to believe that it could be ATP some
how copurified with the protein. Refinement with ATP, ADP+acetate, did
not vary too much in the B factors. The main concern is, only one xtal
was observed at this state ( mimicked like a ATP bound state and
changed conformation and space group). All other xtals (around 10 xtals
were measured) from this plate did not have this change.So it was little
tough to understand that only this xtal has ATP instead of ADP. We were
not able to reproduce xtals of similar space group with similar setup
(with ADP). After tremendous trials we could grow one xtal with ATP
which has the P212121 space group and all the observed changes. Is any
one observed ATP mimicking by compounds other than aluminium/berillyium
Fluoride?
Thanks
Saravanan
Dear All,
Sorry for the little bit off topic. Is there a possibility for covalent
bond formation between beta phosphate of ADP and acetate molecule both
are coordinated by divalent metal ions? I am working on a Kinase
structure which was crystallized with ADP and in presence of 1M sodium
acetate. We observed additional density around ADP that fits perfectly
like a gamma phosphate , mimicking like ATP bound state, surrounded and
coordinated by two metal ions(resolution is 1.4A). There is a change in
space group (from I212121 to P212121 ) and further important
conformation changes are observed around ATP binding pocket and distant
region. This is the only xtal we obtained in this space group, and all
other xtals(measured 10 xtals) from the same plate belong to I212121.
Thanks your help and time!
Saravanan
Replies:
Hello Saravanan
At 1.4 Angstrom resolution wouldn't that suggest that you've somehow got
ATP in there ? I don't think I understand the other option - were you
proposing a ADP-O-C(O)2 arrangement to explain the density ? Surely
that has a rather different shape, considerably different scattering
power at the center of the terminal group (C vs P) and probably
different X-O bond lengths. All of these should show in the density
maps at 1.4 Å, although the bond length issue could be quite subtle.
Phil Jeffrey
Princeton
Perhaps it is ATP?
Mark J van Raaij
Remember that 2xADP can disproportionate into ATP + AMP
Hi Saravanan
It sounds to me like you have ATP there, even though you added ADP. Is
your kinase an inactive mutant? I know kinases can bring nucleotides
along for the ride during purification.
Regards
Christine
--
Dr. Saravanan Panneerselvam PhD
P11 Beamline Scientist
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