[phenixbb] bad density for part of the electron density map
wei.shi118 at gmail.com
Tue Oct 8 07:58:59 PDT 2013
Hi Professor Read,
Thank you so much for your detailed descriptions! I will try it.
On Mon, Oct 7, 2013 at 9:18 AM, Randy Read <rjr27 at cam.ac.uk> wrote:
> It's not necessary to redefine the C-terminal domain with the output PDB
> file. You can just go back to the Configure tab in the Phaser-MR GUI,
> choose the "Input and general options" tab and, where it says, "Use partial
> solution from previous job", choose the job number corresponding to the
> search for the C-terminal domain in the pull-down. The GUI stores data
> about partial solutions and, as long as you provide the same ensemble with
> the same name that was used for the search, Phaser will apply the rotation
> and/or translation that was determined in the earlier job, for any
> components placed in the partial solution. If you hadn't already defined
> an ensemble for the N-terminal domain, you would need to do that now and
> then change the choice of Search ensemble in the Search procedure tab from
> the C-terminal to the N-terminal domain before running the next job.
> One important trick to remember: the list of partial solutions in the
> pull-down menu is not automatically updated, so if you don't see the
> desired job number, click on the refresh button to the right!
> The process is similar for following a separate brute-force rotation
> search with a separate translation search. When you ran the brute-force
> search, you chose "Brute rotation function" for Phaser mode. To run a
> translation search using the orientations found in that search, choose the
> "Translation function" mode. The output rotation list from the brute
> rotation search is in a hidden file (which is probably why you didn't find
> any output), but if you choose the corresponding run from the list of
> partial solutions, then the list will be provided to Phaser for the
> translation search. (If you're used to the CCP4 interface, this is
> different because in ccp4i there's a .rlist file that contains the list of
> orientations.) After the translation search you will want to follow the
> same steps, manually, that the automated mode would use, i.e. packing then
> refinement&phasing. At each step, refresh the list of partial solutions
> and choose the one from the last job.
> There's actually an easier way to carry out a search that uses
> orientations around a known orientation! You can leave the mode as
> "Automated molecular replacement", so the translation, packing and
> refinement steps will still be carried out automatically, but configure
> Phaser so that it uses the brute-force rotation with the rotate around
> option. Click on "Other settings", and make sure that the user level is
> set to Advanced or higher. In the "Rotation peak selection" section, set
> "Select peaks by" to "All", and uncheck "Select clustered peaks". In the
> "Brute rotation function" section, set Volume to "Around", set the Euler
> angles to the expected orientation of the molecule you're searching for,
> and set the Range to something sensible like 30. Further down the window,
> set "Fast rotation target function" to "brute", and then it's ready to run.
> Good luck!
> Randy Read
> On 6 Oct 2013, at 21:39, Wei Shi <wei.shi118 at gmail.com> wrote:
> 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.
>> phenixbb mailing list
>> phenixbb at phenix-online.org
> phenixbb mailing list
> phenixbb at phenix-online.org
> 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
> phenixbb mailing list
> phenixbb at phenix-online.org
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