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).

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.

From: CCP4 bulletin board <[email protected]> on behalf of Jiang Xu <[email protected]>
Sent: Tuesday, June 22, 2021 6:47:36 AM
To: [email protected] <[email protected]>
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?

Thank you,

Best,

Jiang Xu

Lin Chen's Research Group

University of Southern California

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