While working with a low-affinity, nonspecific DNA binding protein, I
ran into this problem dishearteningly often. In several cases, even
when running washed crystals out on a gel had told me that they
contained both DNA and protein (32P-labeling for DNA, Coomassie for
protein), I was unable to obtain convincing difference density for
nucleic acid. As others have suggested, this could be caused by either
low occupancy or multiple binding registers in the same crystal. One
way to overcome the affinity issue might be backsoaking the crystals
into a lower salt concentration, though this method was never
successful for me. You could also continue your screening efforts and
cross your fingers for a crystal form where the DNA is better ordered.
In the end, I developed a screen ("FASTDXL") based on Greg Verdine's
protein-DNA disulfide crosslinking chemistry to tether the nucleic
acid outside the binding site, thereby greatly increasing the local
concentration and fixing DNA in a unique register (Structure 2007,
15(7):773-80; NSMB in press). If you decide to pursue something along
those lines and have success obtaining a structure, be sure to
rigorously validate its biological relevance. Feel free to send me an
email if you have any questions about FASTDXL.
Jacob
Jacob Corn
The Berger Lab
UC Berkeley - Molecular and Cell Biology
[EMAIL PROTECTED]
phone: 510-643-9491
fax: 510-643-9290
Melody Lin wrote:
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
