In addition to excellent Kay's reply.. Also make sure to check refined B factors. Note, if the ligand is not there then that volume is likely filled with bulk-solvent. Now low occupancy in combination with very large B factors may approximate bulk-solvent quite well. The Polder map along with three CC values that its calculation procedure reports should give you the answer whether the ligand is there or not.
Pavel On Wed, Feb 8, 2017 at 2:43 AM, Kay Diederichs < kay.diederi...@uni-konstanz.de> wrote: > 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 >
