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

Wei Shi wei.shi118 at gmail.com
Sun Oct 6 13:39:45 PDT 2013

Hi Professor Read,
Thank you so much for your suggestions!
I used the brute rotation function in phenix GUI (Phaser-MR) but didn't get
any output and I don't know whether I set the running parameters right....
Below is what I did.
I first did the molecular replacement solution using only the C-terminal
domain as search model and got a solution. And, then I loaded the pdb file
from this molecular replacement solution onto Phaser-MR as Ensemble 1 and
click Ensemble is fixed partial solution and also load the pdb file for the
N-terminal DNA binding domain as Ensemble 2 and tell the program to search
for 2 copies of the N-terminal DNA binding domain. And for phaser mode, I
chose Brute rotation function. And then I went to 'settings' and 'search
parameters' and entered rotate, it displays Volume, Euler and Range and I
select 'around' for Volume and for Euler I entered the Euler values from
the molecular replacement solution using C-terminal domain as search model,
and for range I entered 30 (are these values I entered right???). And, I
also unclick 'select clustered peak'. Then, I ran the Phaser-MR with the
above settings but got no output file.... I don't know whether I did
something not correct....
Besides, from the Brute rotation function, what kind of output I am
expecting? How can this be used as input for translational searches and
what are the settings for the run? Thank you so much!


On Fri, Oct 4, 2013 at 4:04 AM, Randy Read <rjr27 at cam.ac.uk> wrote:

> 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
> >
> > _______________________________________________
> > phenixbb mailing list
> > phenixbb at phenix-online.org
> > http://phenix-online.org/mailman/listinfo/phenixbb
> ------
> Randy J. Read
> Department of Haematology, University of Cambridge
> Cambridge Institute for Medical Research      Tel: + 44 1223 336500
> Wellcome Trust/MRC Building                   Fax: + 44 1223 336827
> Hills Road                                    E-mail: rjr27 at cam.ac.uk
> Cambridge CB2 0XY, U.K.                       www-structmed.cimr.cam.ac.uk
> _______________________________________________
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