Distinguish Ca from Mg

From the CCP4bb

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' opinions. The question in my mind is whether the bonding distance with the coordinating oxygens is a discriminator (~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).

> 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' opinions. The question in my mind is whether the bonding distance with the coordinating oxygens is a discriminator (~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 program of your choice!Crystallographically

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 a lot 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 a lot 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.htmlturned 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.