[phenixbb] simulated annealing omit map

Ivan IVANOV ivanov at embl.fr
Thu Nov 13 22:40:30 PST 2014

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

     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, 

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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