[phenixbb] bad density for part of the electron density map

Randy Read rjr27 at cam.ac.uk
Fri Oct 4 01:04:21 PDT 2013

If the density is bad for one domain, it could be poorly ordered (in which case you will have a hard time improving things much), but the high R-factors might be indicating that it's well-ordered but just not placed correctly.  You mentioned that there is a conformational change on binding.  Could this involve a movement of the N-terminal domain, which might not have been modelled correctly yet?

What I would try in this situation is to trim off the N-terminal domain, provide Phaser with the rest of the structure as a fixed partial molecular replacement solution, and then run a search looking for the N-terminal domain.  If it is relatively small, then the signal may be poor so a default search may not work.  Often, with small substituents, the weakest part of the search is the rotation search.  However, you can take advantage of what you know from the apo-structure and assume that the domain will only differ by a relatively small angle (say, up to 30 degrees), generate all orientation angles similar to the orientation of the rest of the protein (achieved by running a brute rotation search around the angles for the rest of the protein, turning off clustering and accepting all rotations), and use this set of orientations for translation searches.

An alternative that might work for small changes in orientation would be to carry out a rigid-body refinement of the N-terminal domain and the rest of the structure as two rigid groups, either in Phaser or in phenix.refine.

Good luck!

Randy Read

On 4 Oct 2013, at 04:51, Wei Shi <wei.shi118 at gmail.com> wrote:

> Hi all, 
> I am working with a dataset in space P212121, resolution 2.8 amstrong, total completeness of the data 96.2% (98.1%), I/sigma 5.2 (3.1). 
> This is a structure of a transcriptional factor (dimer) with the ligand. Upon ligand binding, there is conformational change in some part of the protein and I used the apo protein structure as a search model, and get a molecular replacement solution. After some rounds of refinement and rebuild (mainly in a region in the C-terminal ligand binding domain), the best refinement I have is as follows. But the electron density map for the C-terminal DNA binding (about 80 residues) is still very bad.... I tried to mutate them to alanine and do refinement and also tried to delete the whole region to do refinement, but both of the strategies didn't give me better density which I could use to rebuild the residues manually. I am wondering whether any of you have any ideas about what might go wrong and any suggestions about what to check or try next. Thank you so much! 
>                          start         final
>   ---------------------------------------
>   R-work:           0.3220        0.3100
>   R-free:           0.3916        0.3884
>   RMS(angles):      2.50          1.39
>   RMS(bonds):       0.016        0.010
>                  Ramachandran outliers:   1.8% (Goal: < 0.2%)
>                   Ramachandran favored:  89.0% (Goal: > 98%)
>                       Rotamer outliers:   5.4% (Goal: 1%)
>                        C-beta outliers:   0    (Goal: 0)
>                             Clashscore:  11.50
>                          Overall score:   2.71
> Best,
> Wei 
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Randy J. Read
Department of Haematology, University of Cambridge
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