Tryptone and/or yeast extract is loaded with metals, including zinc. It
is almost impossible to keep zinc contamination at bay without using
carefully defined media and taking heroic measures. (I know this because
of the difficulty of making overexpressed metallo-substituted
zinc-enzymes when this is the only way to do it). So, yes, it is very
possible to observe zinc coordination in heterologously expressed
proteins when exogenous zinc has not be explicitly included.
I don't put that much stock in the sigma level. It is too variable and
depends on the resolution, the current state of the maps (initial or
nearly final phases), and occupancy. You should have very obvious
positive difference density, however. Some clues to Zn-coordination
include the following: (1) Zn(II) is often in a tetrahedral or
pseudo-tetrahedral geometry, and typically accepts a mixture of hard to
soft ligands; (2) Zn-S bonds are approximately 2.3 A; Zn-O or Zn-N bonds
are approximately 2.0 A. If this is compatible with your unconstrained
fit of the data to the density using a Zn(II) ion, then there is a
strong possibility of Zn-coordination.
To confirm Zn coordination, you can measure the Zn content of the
protein sample using ICP-OES, ICP-MS, or TXRF. For ICP-OES, I usually
dilute a protein sample at 10-50 mg/mL 1:50 to 1:150 and analyze a 1.5
mL sample. You can do multiple metals at the same time, including Zn. I
usually also look for Fe, Ni, Cu, Mn and do Co as a control (Co should
be zero or nearly so.) Once you measure metal content, you can estimate
occupancy by measuring the Zn/protein molar ratio. If you do this, use a
non-idiosyncratic (sequence-insensitive) protein assay. I would
recommend a UV-based method using A210 in 0.01% detergent (e.g. Triton
or Brij) For homogeneous proteins, the A210 method is usually accurate
within 5%, which is sufficient to get usable metal:protein ratios. If
you must use a colorimetric method, the only one I trust not to be
idiosyncratic is the microbiuret method, which is as old as dirt. It
usually agrees with the A210 method.
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 11/7/2012 12:29 PM, SD Y wrote:
Dear all,
I have a related question to the one I have posted "low resolution and
SG", on which I am still working based on the suggestions I have got.
The model I have used, has Zn co-ordinated well in tetrahydral fashion
by 3 cys and 1 His residues. They have add Zn in to their experiment.
In my 3.4 A structure (I am still working on right SG), initial maps
show very strong positive density (sigma=6.5) at the place of Zn
( https://www.dropbox.com/s/4jd6gdor87ab9lj/Zn-coordination.png). I
have not used Zn in my experiment. I could only suspect Tryptone and
yeast extract which I used to make media.
I would like to know how likely this positive density belongs to Zn?
How to reason the presence of Zn when its not been used?
Is there is any way to confirm if its Zn. If this is not Zn, what else
could it be? Any thing I could try to rule out or in Zn or other ions.
I appreciate your help and suggestions.
Sincerely,
SDY
