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-------- Original message --------
From: Clemens Vonrhein <vonrh...@globalphasing.com>
Date: 2017-06-16 4:43 AM (GMT-06:00)
To: CCP4BB@JISCMAIL.AC.UK
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.
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.
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.
Cheers
Clemens
[1]
https://www.globalphasing.com/buster/wiki/index.cgi?AutoBusterExampleFormfactor
(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/<http://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 <pafon...@gmail.com> 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 <mohammad.rah...@uef.fi>
> > 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
> >>
> >>
--
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* Global Phasing Ltd., Sheraton House, Castle Park
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