Re: Mg2+ Ca2+

["Eleanor J. Dodson" <ccp4@ysbl.york.ac.uk>]

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"David E. Timm" wrote:
> 
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> Hello,
> 
> We have recently put alot of effort into identifying divalent cations
> present in the active site of an enzyme that was not previously known to
> utilize a metal cofactor.  We had 150 mM Ni2+ as a crystallization additive,
> but the protein had also seen Ca2+ during a thrombin digestion step.  The
> active site actually ended up with a Ca2+ and a Mg2+, with a single Ni2+
> making a lattice contact.  We were helped out by having several data sets
> ranging from 1.25 to 1.9 A, compared to the 2.2 A data trying to distinguish
> between Ca2+ and Mg2+.  I don't have anything to add beyond the previously
> suggested use of anomalous data and a comparison of B-factors for the metal
> ligands, rather than to the overall B.  However, I was surprised that
> difference maps weren't good indicators at 2.2 A.  At 1.9 A we observed
> respective peaks or troughs in the Fo-Fc maps when too light or too heavy a
> cation was used in the model, even though the B-factors were soaking up alot
> of the error.



 Interesting point - could it be that the diffraction ripples from
heavier atoms are more serious at one resolution rather than another -
we had a horrible problem with the bound Ua in Oppa, and I always meant
to follow it up with a careful analysis of diffraction ripple effects at
different resolutions, and never did..
 > 
> With regard to Eleanor's comments about VDW radii, what is the best way to
> handle metal centers in REFMAC to assess coordination geometry based on
> diffraction data rather than refinement restraints?  Is the suggestion to
> just artificially set the metal VDW radius to near zero and see where the
> surrounding ligands end up?  How is the VDWR altered in PROTIN for atom
> types not defined by an ICODE number (VDWR <nvdw> <type_1> <icode_1>
> <dvdw_1> ... ?  Only 10 ICODES are listed in the protin.doc.


 In PROTIN the VDW radii is a function of the ICODE number, and all
metals have the same (7 ). So if you have both a Ca Fe and Mg in your
structure you might need to edit the dictionary to give them different
numbers, then set different VDW radii for each. 1, 2, 3, 4 are C N O and
S; the others can be manipulated to suit your problem..

 Bumping restraints are only imposed if the atom seperation is less than
(VDWR_atom1+VDWR_atom2 -0.5) A  so setting one VDW radius to 0.00
effectively disables this restraint for all possible contacts.
Alternatively you can set a "SPEC DIST" restraint between likely bonding
pairs eg ZN - Ne1 with a large SU. This means that REFMAC will monitor
the distance for you, but again it will not really be restrained..


> Finally, one contributor to the discussion stated that, "Mg2+ should be
> always octahedrally coordinated and an average Mg2+ to O distance of 2.1 A.
> Ca2+ has preferably seven or eight ligand atoms with an average Ca2+ to O
> distance of 2.4 A."  It is dangerous to say "always" in any scientific
> discussion.  We have refined a 1.25 A structure containing a Ca2+
> coordinated by six ligands and a Mg2+ coordinated by five ligands,
> consistent with our ICP Atomic Emission Spectroscopy measurements.  A search
> of the database at http://metallo.scripps.edu/current/raw.html turned up 43,
> 54 and 269 respective matches for Mg2+ coordinated respectively by 4, 5 or 6
> ligands, while 102, 160, 369, 445 and 114 respective matches were made for
> Ca2+ coordinated by 4, 5, 6, 7 or 8 ligands.  Clearly, there is considerable
> variability in metal ion coordination geomtries.
> 


 I am not sure how much value these metal data bases have - certainly
anything taken from the PDB is worse than useless unless you knowexactly
how the refinement was carried out, and consulting PDB entries to
provide a set of likely restraints is not a good idea!!

 There is work going on with the small molecule data base - CSD.

 Marjorie Harding has publidshed something in Acta Cryst D recently
Eleanor