Hi, >(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?
If your DNA density is becoming poor as your refine, it is quite likely that the initial DNA density you are observing after MR is model bias due to inclusion of a DNA model with the protein search molecule. If your protein is small compared to DNA ligand, or your DNA comprises more of the asu content that the protein, you could try doing MR with only DNA search model. If you then get the same solution, that would imply that the DNA density is real. If you have confirmed the presence of DNA in the crystals by silver staining gel, and assuming the crystals were washed well to avoid any DNA sticking to the crystals, etc., then loss of DNA density may indicate that the DNA is not well ordered. >(2) are simulated annealing or cycles of coordinate/B factor refinement enough >to get rid of model bias? Your observation of DNA density becoming poor and/or disappearing probably answers your question.........however, it is difficult to get rid of model bias. Best way to do it is to remove DNA portion from the search model. You may also try composite omit maps and simulated annealing omit maps in CNS which basically calculate maps by removing portions of the model, or prime and switch phasing using RESOLVE to reduce model bias. >(3) Does weak DNA density have to do with data processing? Unlikely, especially if density for your protein is very good. 2.4-2.6A should be quite good to see good DNA density. You might see some differences based on what you mention about including even weak and incomplete high resolution data, but should not result in your overall observation of DNA density becoming poor after refinement. If you are very sure you have DNA in the crystal as protein-DNA complex and you are unable to see DNA density, you might consider alterations in the DNA sequence used in the crystallizations to get a more rigid DNA duplex (assuming you are working with dsDNA), getting more GC bp into it. And also consider introducing Cys mutations into the protein to get a thiol cross-linked protein-DNA complex. Regards, Debanu. -- Debanu Das, SSRL. -----Original Message----- From: CCP4 bulletin board on behalf of Melody Lin Sent: Tue 11/6/2007 7:36 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] elusive DNA density 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
