Print

Print
On Mon, Apr 21, 2014 at 3:36 PM, Faisal Tarique <[log in to unmask]> wrote:
Just in the continuation of my previous mail i again want to ask few question on the metalloprotiens..Apart from factors like occupancy, B factor, coordination sphere and metal ion-ligand distances to distinguish Mg or calcium, can anomalous signal  tell the identity and the type of metal ion bound to the protein,  specifically in the case of Mg and Calcium.

Short answer: if you see a peak in the anomalous difference map, it's almost certainly calcium, but if you don't see a peak, you still can't rule out calcium.

Longer answer: magnesium almost never has observable anomalous signal at the wavelengths we normally use for data collection.  The exception is if you collect extremely redundant data; Wayne Hendrickson has a very convincing example of this (I saw it in a talk, but I'll see if I can find a reference).  Calcium anomalous signal depends on the data quality, but with good data and full occupancy it can show up in the anomalous difference map even at the SeMet K edge (~0.9794Å).  However, this is not guaranteed, especially if it's not very tightly bound.  At 2.6Å resolution it may be more difficult to distinguish, especially if you have other stronger anomalous scatterers.  Collecting very redundant data will help a lot.

.An anomalous data analyzed through Xtriage (phenix) gives a signal of 0.097 with Magnesium while the same gives a signal of 0.1062 with Caclium ( both data sets showing Anomalous flag as true )..can anybody shed some light on which is more true ??

I don't understand this - what exactly is the difference between the datasets?  Anyway, that number is really not intended to be interpreted this way.
 
the data has maximum resolution of 2.6A and i had kept Mg atom at the active site (  protein was incubated with 5mM MgCl2)..just because it is not matching a typical octahedral geometry and exact metal ion-oxygen distance as represented by Cambridge structural database (CSD) my reviewer has asked me to check anomalous signal for both Mg and Ca and ( he is expecting that scattering metal ion it to be Ca ) give appropriate reason for putting Mg there..please give suggestions.

In addition to the anomalous maps, check the difference map (Fo-Fc) and B-factors after refinement with either element at full occupancy.  If it is correctly identified, the difference map should be relatively flat and the B-factor should be similar to the coordinating atoms.  Negative difference map peaks and/or a high B-factor suggest that the element is too heavy; positive peaks and/or low B-factors indicate the opposite.

-Nat