Hello,Thank you for your kind reply.We used glycerol to fish out the
crystals.Sorry for not including more info before, I thinkI should have
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.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 gain.Best
regards,Samer
On Monday, July 20, 2020, 05:39:23 PM GMT+1, Nils Marechal
<[email protected]> wrote:
Dear Samer,
What king of cryo-protectant did you use ?
Such a bent density, with that size, looks like an ethylene-diol.
Best regards,
Nils Marechal De : "samer halabi"
<[email protected]>
A : [email protected]
Envoyé: lundi 20 Juillet 2020 18:17
Objet : [ccp4bb] electron density close Histidine side chain
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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