Dear All, Would you mind if I disrupt your conversation about the omit map or composite omit map ?? I would like to share with you I'm in the same situation as Bishal. I want to see a ligand in the structure I have. I am using Phenix for refinement. When I do a molecular replacement with the model or part of the model, it looks like there is continues density of the ligand on 2Fo-Fc and Fo-Fc map at lower counter level. 1st QUESTION: Once we got the phases from the model which serves for MR, what is the counter level limit for a 2Fo-Fc and Fo-Fc maps we can look at ?? Long time ago I saw some papers in the literature telling about a sigma of 0.4-0.5 there is still useful information. (This first map after MR is not biased by ligand I want to see). Further going on the structure refinement, at some point I add the compound I poorly see and I do 3 different refinements !! One with compound occupancy 0.8, another one with 0.7 and another one with 0.6. Then the density for the compound after refinement appears. At that point I am on the second option in Pavel's suggestions below - I get probably a biased positive density in the map. BUT, I would like to notice that my Fwork and Ffree go significantly down. 2nd QUESTION: Does it mean the compound is there if statistics do down?? About first Pavel's suggestion below when I am doing refinement in absence of the ligand it is the same situation Fwork and Free go down and the ligand density it looks like to be still there and it is NOT completely masked by bulk solvent (but I have to go still to lower counter lever for the 2Fo-Fc map). Another point I would like to arise. I've done a composite omit map with Phenix (because as I told you I like Phenix). One thing it is worth noticing when you are in a hurry or you want to see quick result because you are not patient enough, do not use Phenix for omit maps (it is slow :-) :-) :-) ). Except that I like very much Phenix. In comparison I did also the same maps with CCP4 and at the end it does not seem to change that much the result when you compare both omit maps coming from Phenix or CCP4. About my case the omit map (no biased) gives the same quality of density for the compound as 2Fo-Fc map after MR. I have to go in a lower counter lever to see something. 3rd QUESTION: What is the limit for a composite omit map to look at ?? 0.5-0.6 sigma ?? 4th QUESTION: Do you think I can improve the quality of the density if I burn my crystals (collect several multiplicity) ?? or probably murge different crystals data?? I like very much Dale's offer about the discovery map. I believe there is something there. Sorry for the long mail, Kind regards, Ivan On Thu, 2014-11-13 at 08:38 -0800, Pavel Afonine wrote:
Hello Bishal,
as discussed before, computing ligand-omit map is tricky.
Quoting my previous email:
""" There are two commonly used options for omitting the ligand in order to demonstrate its presence/absence in Fo-Fc OMIT map:
1) Physically remove the ligand from PDB file. Then do some refinement and calculate Fo-Fc map.
2) Keep ligand in the file, set its occupancy to zero. Then, again, do some refinement and calculate Fo-Fc map. In this case you may want to ask refinement program to not move the ligand or move it only a little.
Now, here is why these two options are poor and will not give you what you want.
In the first case the bulk-solvent mask will be set in the ligand region and therefore it will mask ligand density (bulk-solvent will be filled into the ligand region). Depending on the strength of ligand density it may be masked completely or deteriorated.
If you follow the second option you will always get positive density in ligand area. This density may correspond to bulk-solvent, ligand or mixture of both. That is there will be no simple way to differentiate whether this density arises from the ligand or bulk-solvent. """
Dale Tronrud offered a great alternative option:
""" An alternative you might want to consider is what I call the "discovery map". At some point in the refinement process there was a map that convinced YOU that this ligand was present. You should be the hardest person to be convinced so that map will be both an omit map (because the model had been refined without the ligand prior to this) and clear enough to satisfy the reader. """
Now, recently I improved composite OMIT map calculation in
phenix.composite_omit_map
tool, see picture that illustrates how it works:
https://www.dropbox.com/s/dbp8e0348v5p78h/fig_6.png?dl=0
I think at present that's the best option to follow if for some reason you cannot follow Dale's suggestion.
Pavel
On 11/13/14 6:58 AM, Singh, Bishal wrote:
Hello everybody, I am generating figures showing the electron density map around the ligands. I deleted the ligands from final co-ordinate file and then performed refinement with identical parameters as before while keeping simulated annealing=true. I shall be thankful if someone could suggest me whether I consider mFo-DFc map or 2mFo-DFc map or both. Kindly also tell me the minimum acceptable contour level required for showing difference map. All structures are at 1.4 - 2.0 angstrom resolutions.
Regards, Bishal
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