Hi,
If you have a crystal you could do a x-ray fluorescence scan to see what
might be in your crystal and collect anomalous data at higher wavelength
close to the manganese edge to see if it is manganese. These enzymes can
also deal with Zinc which is a bit heavier and might be still bound from
the expression system during protein synthesis.
You could also do a ICP-AAS analysis with your protein and see what you
find if you don't have a crystal anymore. There is also PIXIE for such
an analysis on some synchrotrons available.
There are probably many more options to identify the metal ion.
Christian
Am 09.07.2015 um 10:35 schrieb Dilip Kumar:
> Dear All
>
> I have solved a structure of a metal-ion dependent exonuclease enzyme.
> In homologous structures, two or three Manganese ions are present at
> catalytic center. However, I have used 2 mM MgCl2 in protein
> purification buffer. I tried to fit both of these metal ions at
> catalytic center but in both cases it still shows green density (Sigma
> level ~ 7) in difference map and low b-factor (<10) for these metal
> ions. For better understanding I have attached the screenshot of metal
> ions with difference map on. Please suggest me the possible reasons or
> methods to validate the presence of any other metal ions at catalytic
> center.
>
> Thanks in advance.
>
> Regards
> Dilip Kumar
> Research Associate
> Chemical and Systems Biology Unit
> CSIR-Institute of Genomics & Integrative Biology
> Delhi-110025
|