Engin, Thanks for your help. Initially, I tried the order you are suggesting for the links ("inner first, outer second"), but Phenix quits with the following output: Sorry: Traceback (most recent call last): File "/jcsg/software/phenix/phenix-1.7/phenix-1.7-650/cctbx_project/mmtbx/monomer_library/pdb_interpretation.py", line 972, in apply_mod mod_mon = self.monomer.apply_mod(mod_mod_id) File "/jcsg/software/phenix/phenix-1.7/phenix-1.7-650/cctbx_project/mmtbx/monomer_library/cif_types.py", line 387, in apply_mod result.delete_atom_in_place(mod_atom.atom_id) File "/jcsg/software/phenix/phenix-1.7/phenix-1.7-650/cctbx_project/mmtbx/monomer_library/cif_types.py", line 304, in delete_atom_in_place raise RuntimeError("delete_atom_in_place: unknown atom_id: %s" % atom_id) RuntimeError: delete_atom_in_place: unknown atom_id: O1 apply_mod failure: pdbres="ASN A 91 " comp id: ASN mod id: DEL-O1 This sounded to me like it was trying to delete O1 from my Asn instead of the GlcNAc, which I figured meant that I should reverse the order (i.e., outer residue first, inner residue second). When I did this, phenix completed refinement with no obvious problems in the log file. I initially took this to mean that all was well, until I looked at my coordinates. As for glycans in the same chain or different chains, I agree with you completely. I actually split them into separate chains to try to get around another apparent problem that was cropping up near the top of my log file: Chain: "C" Number of atoms: 5065 Number of conformers: 2 Conformer: "A" Number of residues, atoms: 327, 5018 Unusual residues: {'MAN%DEL-HO3': 1, 'MAN%DEL-HO4%DEL-O1': 1, 'NAG%DEL-O1': 1, 'NAG%DEL-HO4%DEL-O1': 1} Classifications: {'undetermined': 4, 'peptide': 323} Modifications used: {'DEL-HD22': 1, 'DEL-HO3': 1, 'DEL-O1': 3, 'DEL-HO4': 2} Link IDs: {'PTRANS': 18, None: 4, 'TRANS': 304} Not linked: pdbres="NAG C 401 " pdbres="NAG C 402 " Not linked: pdbres="NAG C 402 " pdbres="BMA C 403 " Not linked: pdbres="BMA C 403 " pdbres="MAN C 404 " Unresolved non-hydrogen bonds: 1 Unresolved non-hydrogen angles: 2 Unresolved non-hydrogen chiralities: 1 These "Not linked" sections went away when I moved the glycans to separate chains, so I figured that Phenix might only allow one polymer type in a given chain (not sure if this is true or not). As for the monomer cif files, I don't see an major differences between the cifs from Phenix and those from CCP4 that I have used previously for refinement in Refmac, but I will look into this further. Best, Damian On Feb 28, 2011, at 11:44 PM, Engin Özkan wrote:
Also, I just remembered that Phenix/monomer library may still not have BMA and MAN right. You may have to provide MAN and/or BMA cif files with the correct stereochemistries. Monomer library does not necessarily follow the standards of the PDB chemical component library (but obviously, PDB enforces them).
Finally, you can always check the .geo file that phenix.refine produces, and you will find all the applied restraints. That should definitively tell you your problem.
Engin
On 2/28/11 10:36 PM, Engin Özkan wrote:
Dear Damien,
Let me make a guess: You have the BETA1-4 linkages in the wrong order. If you look in the mon_lib_list.cif file, you can find the line where it says: BETA1-4 1 O4 2 C1 single 1.439 0.020 which tells me that the residue to be defined (residue_selection_1) ought to be the atom atom connecting with the O4 atom (the inner glycan) and the second residue is the one with the C1 atom (the outer glycan). So try switching residue_selection_1 and _2 for your BETA1-4 linkages. I know you are probably thinking BETA1-4 should go 1 to 4, but that is not how it is defined in the monomer library. By the way, your NAG-ASN linkages do look correct, and you have the correct anomers for beta-mannose (BMA) and alpha-mannose (MAN), which is where most people seem to get stuck.
One stylistic question I have for you is, why are you naming your protein and glycans with different chain IDs; aren't they actually covalently linked? There apparently is no right way for numbering glycan residues, and everybody seems to be do a different thing...
Good luck, Engin
On 2/28/11 6:39 PM, Damian Ekiert wrote:
Sorry to bring up an old topic again but I can't find any documentation of glycan refinement on the Phenix website and previous threads don't seem to answer my question.
I am refining a structure with N-linked glycans. I have inserted several "apply_cif_link" records to my .eff file to define the glycan topologies (see below) and fed in a cif file for BMA. After fiddling for quite some time now, I have managed to get around all the error messages people have described previously to disappear. Yet, 1) none of my linkages appear to be enforced and 2) even the restraints on the individual sugars appear to be unenforced (or at least, insufficiently so). For example, some rings are rendered almost flat, or flipped into boat or otherwise distorted conformations, and glycosidic bonds stretch to>2 A.
No obvious signs of trouble in the log file, running the latest version (1.7, also tried 1.6.4), but my glycans look terrible (despite some of the best density I have ever seen! :-) )
Any suggestions would be appreciated!
Thanks,
Damian Ekiert
From my .eff file:
apply_cif_link { data_link = NAG-ASN residue_selection_1 = chain X and resname NAG and resid 401 residue_selection_2 = chain A and resname ASN and resid 91 } apply_cif_link { data_link = NAG-ASN residue_selection_1 = chain Y and resname NAG and resid 401 residue_selection_2 = chain C and resname ASN and resid 91 } apply_cif_link { data_link = NAG-ASN residue_selection_1 = chain Z and resname NAG and resid 401 residue_selection_2 = chain E and resname ASN and resid 91 } apply_cif_link { data_link = BETA1-4 residue_selection_1 = chain X and resname NAG and resid 402 residue_selection_2 = chain X and resname NAG and resid 401 } apply_cif_link { data_link = BETA1-4 residue_selection_1 = chain Y and resname NAG and resid 402 residue_selection_2 = chain Y and resname NAG and resid 401 } apply_cif_link { data_link = BETA1-4 residue_selection_1 = chain Z and resname NAG and resid 402 residue_selection_2 = chain Z and resname NAG and resid 401 } apply_cif_link { data_link = BETA1-4 residue_selection_1 = chain Y and resname BMA and resid 403 residue_selection_2 = chain Y and resname NAG and resid 402 } apply_cif_link { data_link = ALPHA1-3 residue_selection_1 = chain Y and resname MAN and resid 404 residue_selection_2 = chain Y and resname BMA and resid 403 } _______________________________________________ phenixbb mailing list [email protected] http://phenix-online.org/mailman/listinfo/phenixbb
-- Engin Özkan Post-doctoral Scholar Laboratory of K. Christopher Garcia Howard Hughes Medical Institute Dept of Molecular and Cellular Physiology 279 Campus Drive, Beckman Center B173 Stanford School of Medicine Stanford, CA 94305 ph: (650)-498-7111
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