Hello,Thank you for your kind reply.In this case that could be either Zinc or
Nickel. Sorry, I should've mentioned how I purified the protein complex.
The protein is secreted in HighFive cells, to purify it by its 6xHis tag, I
used Ni sepharose excel beads, eluted with Imidazole and then further purified
with size exclusion. To get rid of the tags and leucine zipper I used to mimic
the transmembrane domain, I subjected the protein to V8 edndoproteinase in 0.1M
Tris pH8.5 (cuts after an exposed Glutamate). Then purified again with Ni
sepharose excel and size exclusion prior to crystallisation. It crystallised in
0.2M Zinc acetate, 0.1M Imidazole pH 6.5, 10% PEG 8K. We used glycerol to fish
the crystals out.I have worked on three other crystals of the similar molecule,
which crsytalized in different conditions, and this is the only one I see such
blobs.
Thank you again.Best regards,Samer
On Monday, July 20, 2020, 08:02:04 PM GMT+1, Roger Rowlett
<[email protected]> wrote:
Almost certainly a metal ion, possibly Ni(2+) if a Ni-NTA column was used for
purification. Ni-N bond lengths are typically around 2.0 A. Additional density
is probably coordinated water molecules, which should have similar Ni-O bond
distances around 1.9 A. It is fairly common to find adventitious metal ions
(zinc, copper, nickel) bound to His residues.
_______________________________________
Roger S. Rowlett
Gordon & Dorothy Kline Professor, Emeritus
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
email: [email protected]
On Mon, Jul 20, 2020 at 12:17 PM samer halabi
<[email protected]> wrote:
Hello all,
I have few blobs in an MHC II structure I am working on, especially opposite to
Histidine as in the accompanying screenshot, that I am confused about.
In the crystal conditions, I have Tris, Imidazole, Acetate, PEG and Glycerol.
Whatever ligand I am fitting in I am getting a clash (overlap -1.029), which
makes me think whether there is a covalent bond forming between Histidine and
other molecule. Perhaps by oxidation.
I would greatly appreciate if you can advice me about it, whether there is some
kind of ligand I can try to fit and if this is something that occurs in some
structures.
Thank you.
Best regards,
Samer
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