On the CCP4 Bulletin Board the following was presented:
Question
I am having troubles in identifying two metal ions in a structure.
I used Mg2+ in sample preparation and Ca2+ in crystallization. The
concentration of Ca2+ is a few folds higher than that of Mg2+. The metal
ions have good octahedral geometry and both Mg2+ and Ca2+ could fit and be
well refined. I am at almost the final stage of refinement with R/R-free
20.5/22.4 (Ca2+) or 20.8/22.6 (Mg2+) for data between 10-2.2Å. Difference
density map doesn't resolve this ambiguity, i.e. both type of ions have
relatively clean maps. I am inclined to the Ca2+ because the crystal only
grew in the presence of this ion. However, one of the metal sites doesn't
involve intermolecular interaction at all, so at least for this site it
should have nothing to do with crystallization.
I would like to know about the experts' opions. The questons in my mind is
whether the bonding distance with the coordinating oxygens is a
discrimnator (~2.3Å in my case)? Also how much the B-factor can tell? (in
my case, Mg2+ ~12, Ca2+ ~29, average of the molecule ~37).
Answers
> The metal ions have good octahedral geometry and both Mg2+ and Ca2+
could fit and be well refined. I am at almost the final stage of refinement with R/R-free
20.5/22.4 (Ca2+) or 20.8/22.6 (Mg2+) for data between 10-2.2Å.
You should be including the low resolution data if you have it (i.e.,
20 - 30Å) and this will allow an accurate bulk solvent correction (this
is really just general advice).
> Difference density map doesn't resolve this ambiguity, i.e. both type of ions have
relatively clean maps.
Because the change in B-factor is mopping up the difference. See
below.
> I would like to know about the experts' opions. The questons in my mind is
whether the bonding distance with the coordinating oxygens is a
discrimnator (~2.3Å in my case)? Also how much the B-factor can tell? (in
my case, Mg2+ ~12, Ca2+ ~29, average of the molecule ~37).
You should check the B-factor of the protein atoms that are the
ligands. If the atoms have B-factors around 12, then the Mg2+ appears
to be appropriate. If they are around 29, then the Ca2+ is likely the
answer.
You could also use Eleanor Dodson's trick of looking at the anomalous
signal in your data (assuming you kept track of it coming out of
scalepack or whatever you used). Ca2+ has a small but detectable
anomalous signal, and if the data are ok, should be visible in an
anomalous fourier.
There are a few things you can do to distinguish between Mg2+ and Ca2+:
Metal-Ligand Geometry
Mg2+ should be always octahedrally coordinated and an average Mg2+ to O
distance of 2.1Å.
Ca2+ has preferably seven or eight ligand atoms with an average Ca2+ to O
distance of 2.4Å.
Both metals are "hard" metals which like "hard" ligands, that is oxygen,
only (nitrogen is a very rare exception, sulfur shouldn't appear). There are
two excellent reviews about metals in proteins: Glusker, J. P. (1991)
Advances in Protein Chemistry, Vol. 42, 1-75, and Harding, M. M. (1999 )
Acta Crystallographica, Vol. D55, 1432-1443.
You have observed an octahedral ligand sphere (indicative for Mg2+) with an
average metal-to-ligand distance of 2.3Å (indicative for Ca2+). However, be
cautious! Your metal-ligand distances are the result after refinement
usually with geometrical restraints, in this case van der Waals radii. In
X-PLOR and CNS, the van der Waals radii of Mg2+ and Ca2+ are by far too
large resulting in too large metal-to-ligand distances (which could be an
explanation for 2.3Å for Mg2+-to-oxygen distance)! I use sigma of 0.8552
for Mg2+ which gives an energy minimum for Mg2+-to-O at 2.08Å, and 1.4254
for Ca2+ which gives an energy minimum for Ca2+-to-O at 2.4Å (look at the
formula for the energy minimum, insert the value for oxygen and solve for
the "best" value for the metal). So, please, before you judge refined
metal-to-ligand distances, check the "ideal" geometry parameter of the
refinement porgram of your choice!
Crystallographically
Ca2+ has 8 more electrons than Mg2+ which should give you a much higher
electron density (contour at, say, ~4 rmsd(rho): do you see the well ordered
sulfurs and your metal, only?). But with this you can only identify Ca2+ if
its occupancy is close to unity. If the occupancies are close to unity, a
falsely placed Mg2+ instead of a real Ca2+ would have a very low B-factor
and vice versa.
Do you still have the raw data? If yes, don't merge the Friedel pairs to
get any anomalous signal in the data. Calculate an anomalous difference
Fourier (CCP4 FFT): do you see the metal? Calcium has an f'' of 1.286
electrons at CuKa radiation, which is high enough, whereas magnesium has
only an f''=0.177, which is neglectable.
If you still have crystals, soak one with Mn2+ instead of Mg2+: Mn2+ is
a very good substitute for Mg2+ but has 13 more electrons. If you calculate
an (Fo(Mn2+)-Fo(unknown)) electron density map, you should see a clear
signal if unknown=Mg2+ and a weak signal (if any) if unknown=Ca2+
I 'd suggest Ca2+ from your bond distances and B-factor (What B-factors do
the ligand atoms have?). Watch out for waters -- Ca2+ is often sevenfold
coordinated while Mg2+ only sixfold, although I have seen Ca2+ ions
with an octahedral ligand sphere. At what wavelength did you collect your
data? CuKalpha would offer a unique possibility by calculating an
imaginary electron density map (coefficients (F+ - F-)exp[i(phimodel -
pi/2)]) which should show nice high peaks on the Ca2+ (and the sulphur
atoms). I think the f'' component for Mg2+ is too small to give a
detectable signal here, so this should discriminate the two alternatives.
However, to do this you must have collected the Friedel pairs ....
Even a little anomalous data will decide this question.
If you run FFT with DANO=D_nat PHI=PHIC you should see very clear peaks
for a Ca and none for Mg.
Everyone collects some anomalous data - but if you run SCALEPACK with
ANOM NO, you can lose it. If you always set ANOM YES, you still get a
merged <I> for all hkl and -h-k-l pairs but the output also preserves
the anomalous differences where observed. This is the default for
SCALA.
Looking at bond distances is rather risky; the refinement programs often
have "hidden" restraints which can distort your geometry. For instance
most programs apply a VDW repulsion unless you specifically request that
it be turned off.
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 1.9Å, compared to the 2.2Å 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 2.2Å. At 1.9Å 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.
Finally, one contributor to the discussion stated that, "Mg2+ should be
always octahedrally coordinated and an average Mg2+ to O distance of 2.1Å.
Ca2+ has preferably seven or eight ligand atoms with an average Ca2+ to O
distance of 2.4Å." It is dangerous to say "always" in any scientific
discussion. We have refined a 1.25Å 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 geometries.