The geometry is difficult to see from a static image, but it looks like
you possibly have a His(2)Glu(4) coordination environment around what
looks like one likely metal ion, based on the difference density. Zn(II)
is typcially (but not always) tetrahedral with Zn-O/N bond distances of
approximately 2.0 A. You should look carefully at the coordination
environment of the putative metal ion to determine if it is tetrahedral,
pentacoordinate (like a trigonal bipyramid), or octahedral. Zn(II) can
adopt all of those coordination environments, but octahedral environment
could be a lot of different metals, e.g., Fe(II), etc. Octahedral bond
lengths for metal-bound ligands will be different from tetrahedral or
pentacoordinate bond lengths. BTW, both Zn and Fe are very common
adventitious metal ions in protein preps, and are very difficult to exclude.
You have options for directly identifying the metal in the home lab: If
the metal is bound tight enough (and it may be stoichiometric) then (1)
ICP-OES or (2) ICP-MS or (3) TXRF of a protein sample should be able to
detect its presence. TXRF is really nice in that there is no prep and it
will work on a few uL of sample, but the others are also quite
sensitive, and will only require 10-50 uL of sample depending on your
protein concentration.
When you are a building a metalloenzyme model you should really have
some solid evidence that a metal ion is present by (1) inclusion in the
crystallization medium, (2) direct determination by an analytical
technique, (3) UV-visible spectroscopy (when appropriate--obviously
Zn(II) is d10 and silent in the visible d-d transition wavelength
range) and/or (4) appropriate coordination geometry and bond lengths.
Cheers,
_______________________________________
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu
On 1/16/2013 2:15 PM, ruisher hu wrote:
Hi, Dear All,
I recently got a dataset about 2.3 A resolution, however, I got some
trouble assigning the metal sites. It suppose to have multiple binding
site(possibly four) around those four glu residues in the center (see
the attached figure), however, it shows up a huge single positive
density ,clustered in the binding center. The signal is pretty strong
and I think zn is definitely there. When I tried to put four zns
around, the geometry doesn't look very good and there is still some
positive density in the center (although get weaker) and the bfactor
of metals are high like 100. Does anyone know what's going on?Does it
mean only one single site in the middle?Or maybe just metals are too
mobile? What's the best way to tell how many metal sites are actually
there?Which experiment can I use to test? Thanks very much.
Best,
R
