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Hi;
You might also consider if it is likely that you have statistical
disorder, causing a blur in the DNA. This could be the case if your
binding is not specific, in which case it is possible that DNA is
shifted by a couple of bases (or if your DNA is "nearly palindromic"
you might have some AsU containing the DNA in one direction and other
in the reverse direction).
Obviously, for this to be the reason of the weak density you observe,
you should first rule out the model bias introduced by MR (as
mentioned by Debanu), an dalso consider what is the sequence of your
DNA, it's structure, and the likeliness that it is not bound to the
same way to the protein in each copy.
Serge.
PS : If you expect the DNA to be making some fibbers in the crystal,
you should definitely be able to see from the diffraction images that
you DNA is present by a strong SF along the fibber axis around the
base stacking distance. This would also be a good proof that you
indeed have diffracting DNA.
Le 7 nov. 07 à 04:36, Melody Lin a écrit :
Dear all,
I've been working on a series of DNA-protein complex structures. In
my recently acquired data sets, I got almost no density for DNA if
I do molecular replacement or rigid body fitting with the protein
structure, although I am 100% sure I have DNA in the structure by
indepenent means. If I use models with DNA, I could find some DNA
density with those data sets, but as I refine the structure, the
density became very poor. The resolutions for those data sets are
between 2.0-2.4 A. Also, if I use the scaled data from synchrotron
rather than the re-scaled data at home, I got better DNA density,
although for re-scaling, I used site parameters that I copied done
from synchrotron. The only differences between those two sets of
scaled data are: (1) the original scaled data take into account all
reflections, including high resolution data with low completeness/
redundancy, which are cut in the re-scaling; (2) error models were
changed so chi squares for each bin are 0.8-1.2 for re-scaling.
My (very naive) questions are: (1) Does the DNA density I saw in
the cases where I use models with DNA for MR/rigid body fitting
only reflect model bias? (2) are simulated annealing or cycles of
coordinate/B factor refinement enough to get rid of model bias? (3)
Does weak DNA density have to do with data processing?
Thanks very much for any suggestion,
Melody Lin
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