[phenixbb] Glycan Links/Restraints

Engin Özkan eozkan at stanford.edu
Tue Mar 1 14:53:52 PST 2011


Dear Damian,

On 3/1/11 2:09 PM, Damian Ekiert wrote:
> 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.
>
You misunderstood me. There is no logic as outer first, inner later. 
Every link description is what it is; you have to carefully read the 
monomer library description file. As I said in my previous email, your 
NAG-ASN linkages were right in your first email, only the BETA1-4s were 
wrong.
> 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).
I never heard of that. And I have refined many structures with multiple 
polymer types in single chains (with versions 1.6.4 and before).
> 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.
That is not surprising. The monomer libraries are common to both. I 
suggest that you check your refmac-refined model for beta-linked BMAs 
and alpha-linked MANs. As a reminder, beta-NAG and beta-MAN (BMA) have 
equitorial linkages at their C1, and alpha-MAN has axial.
> 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
>>>>      }
>>>> _______________________________________________
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>>>> phenixbb at phenix-online.org
>>>> 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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-- 
Engin Özkan
Post-doctoral Scholar
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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