Hi Rob,
I too have encountered this issue occasionally in Phenix.refine & Phenix.real_space_refine.
More often than not in my experience, it comes down to inappropriate addition of a second hydrogen atom to the ASN ND atom.
This makes phenix believe that the NAG is floating near the fully hydrogenated ASN (minus an -OH group) and it doesn't enforce the Asn-NAG bond.
So, my solution:
1.
Build the Glycan in Coot using the Glycan builder tool in the Carbohydrate module (this sets everything up correctly)
2.
If you still need to add hydrogen atoms due to other rebuilding add them in a separate ReadySet run.
3.
Go Back into Coot and make sure that ReadySet hasn't added back the extra hydrogen atoms to the modified ASN(s). Delete them if it has.
4.
Disable the automatic addition of hydrogen atoms in Phenix.refine (I am a GUI user, there is a check box for this, I assume there is also a command line option.)
5.
Run Phenix.refine with the automatic linking options on as you described below.
Hopefully this should work.
Good luck!
Dave
--
Dr David C. Briggs CSci MRSB (he/him)
Principal Laboratory Research Scientist
Signalling and Structural Biology Lab
The Francis Crick Institute
London, UK
Working hours: Mon-Fri 0900-1700
==
about.me/david_briggshttps://about.me/david_briggs | OrcIDhttps://orcid.org/0000-0002-9793-7339 | Google Scholar https://scholar.google.co.uk/citations?user=DRKG5KwAAAAJ
________________________________
From: [email protected]
Sent: 28 June 2024 12:14
To: [email protected]
Subject: [phenixbb] real_space_refine is un-glycosylating my protein
External Sender: Use caution.
Hi,
I am trying to do a refinement with phenix.real_space_refine (RSR) on an EM map with a protein and a known ligand (NAG is the monomer code).
In the pdb file supplied the C1 atom in the NAG molecule is positioned nicely in the EM density at a distance of 1.38Å from a ND2 atom in an ASN residue of the protein. The O1 atom in the NAG molecule is not present suggesting there should be a covalent bond here I believe. Both ASN and the NAG molecule have the same chainID.
The problem is that RSR moves the sugar away even though I have used command line options such as pdb_interpretation.automatic_linking.link_carbohydrates=True or even pdb_interpretation.automatic_linking.link_all=True. The atoms end up at an un-bonded distance of 2.85Å from each other after refinement to the point that one of them is poking out of the density.
Are my phil parameters incorrect? I noticed that I don't get an error message if I misspell say pdb_interpretation.automatic_linking.link_carbohydrates to pdb_interpretation.automatic_linking.link_carbohydrate. RSR still runs to completion so I'm wondering if it actually interprets the PHIL parameter at all.
Another peculiarity is that the log file comments that NAG is an unusual residue rather than stating it is a well-known carbohydrate.
If anyone on here knows how to compel phenix RSR into creating automatic links to carbohydrates suggestions would be much appreciated.
Many thanks,
Rob
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