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

Hi, 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 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!

Best, Wei

On Fri, Oct 4, 2013 at 4:04 AM, Randy Read 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 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 Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills Road E-mail: [email protected] Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk

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------ 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: [email protected] Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk