I agree with Herman about shorter soak. In addition, if your crystals can tolerate any change in pH, you can slow your reaction down by tweaking this parameter. Another thing you should think about is the kinetics of the reaction, especially the rate limiting step. If the rate limiting step is product release, for example, then it may be challenging to capture the initial substrate bound form.
On Wed, Feb 8, 2017 at 7:54 AM, <herman.schreu...@sanofi.com> wrote: > 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 > -- [This e-mail message may contain privileged, confidential and/or proprietary information of H3 Biomedicine. If you believe that it has been sent to you in error, please contact the sender immediately and delete the message including any attachments, without copying, using, or distributing any of the information contained therein. This e-mail message should not be interpreted to include a digital or electronic signature that can be used to authenticate an agreement, contract or other legal document, nor to reflect an intention to be bound to any legally-binding agreement or contract.]
