[phenixbb] [ccp4bb] missing covalent bond between asparagine and N-acetylglucosamine using coot carbohydrate module and phenix.refine

[David Briggs]

Hi Jiang Xu,

(Phenixbb added as I suspect this is Phenix not coot that has caused your issue)

Did you check the "add hydrogens" button in Phenix.refine? Sometimes this can erroneously add a second H to the ND of glycosylated Asn residues. (I assume this is because bond distances in the input PDB file fall outside Phenix's definitions of an Asn-NAG link).

If the second H is added, this breaks the refinement of the Asn-NAG bond.

My work around is to run "Ready Set" to add hydrogens as a separate job, and then manually inspect and correct the Asn residues as necessary before running Phenix.refine and _not_ checking the "add hydrogens" box.

I hope this fixes your problem.

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 at JISCMAIL.AC.UK> on behalf of Jiang Xu <foxjuly at GMAIL.COM>
Sent: Tuesday, June 22, 2021 6:47:36 AM
To: CCP4BB at JISCMAIL.AC.UK <CCP4BB at JISCMAIL.AC.UK>
Subject: [ccp4bb] missing covalent bond between asparagine and N-acetylglucosamine using coot carbohydrate module and phenix.refine


External Sender: Use caution.

Hello guys,
   I have a problem building and refining an xtal structure. My protein has a N-glycosylation site and I want to add N-acetylglucosamine and manose to my protein structure. I used Coot's carbohydrate module and let Coot automatically build the sugar chain to the electron density. It worked pretty well, but I noticed that the bond between the amine group of asparagine and the C1 of N-acetylglucosamine is depicted as a dashed line, rather than a solid line in Coot. After refinement of the structure using Phenix.refine, I found the bond just disappeared, and the amine group still has two hydrogen atoms, which should contain one hydrogen atom.  So,  how to solve this problem?
[image.png]
Thank you,
Best,
Jiang Xu
Lin Chen's Research Group
University of Southern California

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