Dear Petr, Another possibility is that your very good substrate got turned over by the enzyme and that the 5 atoms with good electron density you see is all that is left. When you know the enzymatic reaction, this is easy to check. If this is the case, you should try a short soak (5-30 minutes) and immediately freeze the crystals.
On the other hand, if your difference map shows reasonable density at the 1.5 to 2-sigma level I would not worry too much. Best, Herman -----Ursprüngliche Nachricht----- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Kay Diederichs Gesendet: Mittwoch, 8. Februar 2017 11:43 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] Composit omit map vs. ligand Dear Petr, if I understand correcty, the mFo-DFc density (1) shows almost nothing, but the 2mFo-DFc (2) as well as the composite omit map (3) show the ligand? As you say, the apparent contradiction between (1) versus (2)&(3) is unexpected. One explanation could be that the Fc are simply too bad, i.e. the model not good enough to result in useful signal in the difference map. OTOH, that you see the ligand in (2) may be simply model bias, so is not meaningful. (3) is hopeful since there is no model bias. I would suggest to - refine the occupancy, to find out why the density is so weak - calculate a (Fo,soak - Fo,native) (4) map with phases from a model unbiased by the ligand - try a Polder map (5) - If the occupancy is around 0.5 or higher, that would be a good sign. - but if you don't see density in (4) and (5), then your ligand is probably not there in any useful amount I consider (4) as the most sensible method to show presence of the ligand, and it should convince reviewers. HTH, Kay On Wed, 8 Feb 2017 09:05:50 +0100, Petr Kolenko <petr.kole...@fjfi.cvut.cz> wrote: >Dear colleagues, > >we have a dataset with potential enzyme:ligand complex at 2.2 AA >resolution. The ligand is very good substrate for the enzyme, we used >soaking. We do not see the ligand in the regular difference electron >density, only five out of twenty atoms. However, the ligand placed at >the active site (model used from structure of a mutant variant) is >refined well, giving no negative peaks in difference electron density >map and nice observed electron density. I have calculated composit omit >map with annealing in Phenix (input model did not contain the ligand) >and the electron density for the ligand is there. > >I have my own opinion, but we are desperate to obtain such data (more >than 40 crystals already tested). My question is, would this be proof >of presence of the ligand with reduced occupancy? Will this map >convince the reviewers? Is there any other way to validate presence of the >ligand? > >Best regards, >Petr
