Hi Clemens,
> -----Original Message-----
> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of
> Clemens Vonrhein
> Sent: Friday, June 16, 2017 12:42
> To: [log in to unmask]
> Subject: Re: [ccp4bb] Problem with Mg2+ binding site refinement
>
> Hi,
>
> On Fri, Jun 16, 2017 at 10:47:20AM +0530, Prem Prakash wrote:
> > Hi Mubinur,
> > I got the same situation while refining my protein complex with
> > Lanthanum ion complex, reducing the occupancy while refining solved the
> problem.
>
> That might have not been the whole story through. You still want to use the
> correct formfactor (as Eleanor said) for each atom in your model. The
> formfactors we usually use in refinement (e.g. $CCP4/lib/data/atomsf) are
> for CuKa wavelength - and the data might have been collected at a different
> wavelength. Therefore those formfactors might not be correct in that case.
>
> For most atoms in your model that won't matter that much, since f'
> doesn't change very much with resolution:
>
> 0.8A 0.9A 1.0A 1.1A 1.2A 1.3A 1.4A 1.5A 1.6A 1.7A 1.8A 1.9A
> 2.0A
> ---------------------------------------------------------------------------------------------
> ---
> C : 0.00 0.00 0.00 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.02 0.03
> N : 0.00 0.01 0.01 0.01 0.02 0.02 0.02 0.03 0.03 0.03 0.04 0.04 0.05
> O : 0.01 0.01 0.02 0.02 0.03 0.03 0.04 0.04 0.05 0.05 0.06 0.07 0.07
> P : 0.11 0.13 0.16 0.18 0.21 0.23 0.25 0.27 0.29 0.31 0.33 0.34 0.35
> S : 0.13 0.16 0.19 0.22 0.24 0.27 0.29 0.31 0.33 0.34 0.36 0.37 0.37
>
> (S and P do have a small change though). However, for other atoms that can
> be much more extreme:
>
> 0.8A 0.9A 1.0A 1.1A 1.2A 1.3A 1.4A 1.5A 1.6A 1.7A 1.8A 1.9A
> 2.0A
> ---------------------------------------------------------------------------------------------
> ---
> Br : -1.05 -3.12 -2.23 -1.62 -1.31 -1.11 -0.95 -0.82 -0.71 -0.61 -0.52 -0.45 -
> 0.37
> Cl : 0.15 0.19 0.22 0.25 0.27 0.30 0.32 0.34 0.35 0.36 0.37 0.37 0.37
> Ca : 0.23 0.27 0.30 0.33 0.34 0.35 0.35 0.35 0.32 0.29 0.25 0.19 0.11
> Mg : 0.05 0.06 0.08 0.09 0.11 0.13 0.14 0.16 0.17 0.19 0.20 0.22
> 0.23
> La : -0.47 -0.37 -0.34 -0.38 -0.50 -0.72 -1.04 -1.50 -2.11 -2.93 -4.06 -5.75 -
> 8.32 Se : -0.64 -1.62 -3.48 -1.96 -1.52 -1.26 -1.08 -0.94 -0.81 -0.71 -0.62 -
> 0.53 -0.46
>
> (running CROSSEC will give you those numbers).
>
> When refining against your data at a wavelength different than CuKa, you
> need to adjust your formfactors accordingly (see e.g. [1]). So if you collected
> your Lanthanum dataset at slightly longer wavelengths than CuKa, you
> should tell your refinement program to adjust its scattering factors - and
> since f' is lower, this might already remove that negative density without the
> need to fudge it via a reduction in occupancy. Of course, you could still have a
> partially occupied ion, but with the correct formfactor you will at least have
> the chance to get the occupancy adjustment right.
Exactly. The occupancy column is to model the atomic occupancy, it is not a fudge factor.
> I always thought that the very common approach of adjusting SE atom
> occupancy down to 0.75 or 0.80 in Se-MET (MSE) structures deposited in the
> PDB is not necessarily due to only partial incorporation of Se-MET (versus S-
> MET) during expression, but can sometimes be attributed to using the wrong
> formfactor during refinement.
Yes, we looked at that in PDB-REDO as well. Also hacking the Se occupancy to match the effective scattering is only half the story as the rest of the time there is a sulfur atom rather than nothing.
> Of course, it is very helpful if data processing packages keep the wavelength
> information in the MTZ file correct. Then one can at least compute
> theoretical f' values for a given wavelength. Using measured f' values from a
> good fluorescence scan when collecting data close to the edge is obviously
> even better.
Yes, please. On the PDB-REDO server we get a lot of MTZ files without wavelength and it is not obvious where that problem comes from. In our lab we mostly use Synchrotron -> XDS -> Aimless and that always neatly puts the wavelength in the MTZ file without any particular effort.
Cheers,
Robbie
>
> Cheers
>
> Clemens
>
> [1]
> https://www.globalphasing.com/buster/wiki/index.cgi?AutoBusterExampleF
> ormfactor
> (slightly out-of=date, but gives the idea). Assuming the
> wavelength in the MTZ header is correct (e.g. from autoPROC,
> www.globalphasing.com/autoproc/) and you are away from the edge,
> using AutomaticFormfactorCorrection=yes is all you need to add to
> the command-line. Otherwise one can use
> e.g. FormfactorCorrection="Se:-6.5 Ca:0.27" or such).
>
>
> > Good luck
> > P.P
> >
> > On Thu, Jun 15, 2017 at 11:17 PM, Pavel Afonine <[log in to unmask]>
> wrote:
> >
> > > Hi Mubinur,
> > >
> > > try without "metal restraints" and see if that helps. As others
> > > suggested, make sure 2+ is present in rightmost column of PDB file.
> > > The side may be partially occupied, so refining occupancy of Mg2+ is not a
> bad idea.
> > >
> > > Pavel
> > >
> > > On Wed, Jun 14, 2017 at 2:44 PM, Mohammad Rahman
> > > <[log in to unmask]>
> > > wrote:
> > >
> > >> Dear All,
> > >>
> > >> I am trying to refine a tetrameric enzyme structure that was
> > >> determined at 2.7 Å. The structure contains a Mg2+ binding site in each
> monomer.
> > >> After refinement, in Mg2+ binding site negative density (red) has been
> > >> found as in pictures. I am using Phenix refine, and during
> > >> refinement, metal restrains were used. Herewith I have attached the
> > >> refinement statistics.
> > >>
> > >> please help me to overcome this problem.
> > >>
> > >> Thank you
> > >>
> > >> -Mubinur
> > >>
> > >>
>
> --
>
> *--------------------------------------------------------------
> * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com
> * Global Phasing Ltd., Sheraton House, Castle Park
> * Cambridge CB3 0AX, UK www.globalphasing.com
> *--------------------------------------------------------------
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