Hi Samer, (1) Given that you say there is a relatively high concentration of Zn2+ ions in the crystallisation buffer, I suspect this might be your candidate.
(2) If the Zn2+ ion is correctly represented in the pdb file, your refinement program of choice will know what the target co-ordination distances should be, and should not give clash issues. Good luck, Dave -- Dr David C. Briggs Senior Laboratory Research Scientist Signalling and Structural Biology Lab The Francis Crick Institute London, UK == Diamond User Committee MX representative == about.me/david_briggs ________________________________ From: samer halabi <samer_...@yahoo.com> Sent: 21 July 2020 11:08 To: ccp4bb@jiscmail.ac.uk <ccp4bb@jiscmail.ac.uk>; David Briggs <david.bri...@crick.ac.uk> Subject: Re: [ccp4bb] electron density close Histidine side chain 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 <david.bri...@crick.ac.uk> 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<https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fpmc%2Farticles%2FPMC2872550%2F%23!po%3D0.694444&data=02%7C01%7C%7Cdfc2bafb083e44e4f00e08d82d5e137e%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C1%7C637309229367599545&sdata=u9oHbg6HzNOIeQFMUadyK4OIP1%2BaNFfLAdiPb7OAG8c%3D&reserved=0>) 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 <CCP4BB@JISCMAIL.AC.UK> on behalf of Nils Marechal <00004954a024d277-dmarc-requ...@jiscmail.ac.uk> Sent: Monday, July 20, 2020 5:27:37 PM To: CCP4BB@JISCMAIL.AC.UK <CCP4BB@JISCMAIL.AC.UK> 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" <000030c2162795b2-dmarc-requ...@jiscmail.ac.uk> A : CCP4BB@JISCMAIL.AC.UK 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 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1<https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2FWA-JISC.exe%3FSUBED1%3DCCP4BB%26A%3D1&data=02%7C01%7C%7Cdfc2bafb083e44e4f00e08d82d5e137e%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C1%7C637309229367599545&sdata=5PpyuBq33TvQkS2ky2mUPFUxwLdZwKCKLfJB9thJPTk%3D&reserved=0> ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1<https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2FWA-JISC.exe%3FSUBED1%3DCCP4BB%26A%3D1&data=02%7C01%7C%7Cdfc2bafb083e44e4f00e08d82d5e137e%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C1%7C637309229367609542&sdata=Ug6lq5CwEH5yc5qAYEHwGLv8dEI5fxwTBDPaEXOU8EY%3D&reserved=0> The Francis Crick Institute Limited is a registered charity in England and Wales no. 1140062 and a company registered in England and Wales no. 06885462, with its registered office at 1 Midland Road London NW1 1AT ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1<https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2FWA-JISC.exe%3FSUBED1%3DCCP4BB%26A%3D1&data=02%7C01%7C%7Cdfc2bafb083e44e4f00e08d82d5e137e%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C1%7C637309229367609542&sdata=Ug6lq5CwEH5yc5qAYEHwGLv8dEI5fxwTBDPaEXOU8EY%3D&reserved=0> The Francis Crick Institute Limited is a registered charity in England and Wales no. 1140062 and a company registered in England and Wales no. 06885462, with its registered office at 1 Midland Road London NW1 1AT ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
