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
