Hello,Thank you for your kind reply.If the distances are still less than 2.2Å,
would coot and Refmac still consider that as a clash. When I try to fit in
Imidazole, even if the distance is more than 2.8Å, it is still considering it a
clash.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, 06:25:09 PM GMT+1, David Briggs
<[email protected]> wrote:
Hi Samer,
Did you use a His tag/Ni-NTA during purification? Sometimes Ni2+ ions leach off
the Ni-NTA - maybe the two "ears" are accompanying waters?
Ni-His co-ordination distance is pretty short
(2-2.2Å - table 3
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2872550/#!po=0.694444) and might
account for your bump when you model in a ligand.
Good luck!
Dave
--
Dr David C. Briggs
Senior Laboratory Research Scientist
Signalling and Structural Biology Lab
The Francis Crick Institute
London, UK
==
about.me/david_briggs
From: CCP4 bulletin board <[email protected]> on behalf of Nils Marechal
<[email protected]>
Sent: Monday, July 20, 2020 5:27:37 PM
To: [email protected] <[email protected]>
Subject: Re: [ccp4bb] electron density close Histidine side chain 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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