Re: [ccp4bb] Dealnig with O-linked mannose
Thank you all for helpful suggestions. My question was how to properly connect a mannose to a serine and real-space refine the result. My apologies for not being clear enough. Coot can't find MAN-SER or SER-MAN in it's library (Coot 0.7.1, mon_lib 5.41) It does not automatically make the bond between MAN C1 and OG of SER either. Here is the way I finally made the connection followed by refinement it in Coot: 1) Get monomer... MAN 2) real-space refine MAN into reasonable position 3) Delete hydrogens and reducing hydroxyl Bond is not detected 4) Extensions-Modelling ...-make Link (Click 2 atoms) ... Click on C1 of MAN and OG of SER dashed bond appears 5) Sphere refine A whole different issue that was touched in the replies is how this model will be handled by refinement programs. For the sake of a record, I am using Phenix and it seems to not respect the described link without massaging by means of either pdb.edits or apply_link.def. Also, phenix.refine does not produce LINK records in the output PDB, so step 4 might have to be repeated. Best regards, Dmitry smime.p7s Description: S/MIME cryptographic signature
Re: [ccp4bb] Dealnig with O-linked mannose
Hi, On 11/20/13, 4:17 PM, Zhijie Li wrote: If you need to refine the structure with phenix.refine then you need to make an edit file to specify that the mannose C1 is linked to the ser OG by a covalent bond. I am not sure if this is what was meant, but you don't need to define bonds explicitly in phenix. Phenix.refine has the same (or a forked version, I am not sure) monomer library as refmac's, and it contains the MAN-SER and MAN-THR linkages, which includes bond distance and angle restraints, but no torsion angles (see below). You can invoke a MAN-SER or MAN-THR linkage from the phenix.refine GUI, or add a parameter file to define it. You shouldn't need to specify distances and angles. I am guessing the not-fully mature linkage-detection function in phenix, which works well for N-linked sugars, might also work for O-linked, but Nigel Moriarty from the PHENIX team would know (looking forward to this being automatic). By the way, this is how MAN-SER looks in the latest phenix monomer library: # data_link_MAN-SER # loop_ _chem_link_bond.link_id _chem_link_bond.atom_1_comp_id _chem_link_bond.atom_id_1 _chem_link_bond.atom_2_comp_id _chem_link_bond.atom_id_2 _chem_link_bond.type _chem_link_bond.value_dist _chem_link_bond.value_dist_esd MAN-SER 1 C1 2 OGsingle 1.4390.020 loop_ _chem_link_angle.link_id _chem_link_angle.atom_1_comp_id _chem_link_angle.atom_id_1 _chem_link_angle.atom_2_comp_id _chem_link_angle.atom_id_2 _chem_link_angle.atom_3_comp_id _chem_link_angle.atom_id_3 _chem_link_angle.value_angle _chem_link_angle.value_angle_esd MAN-SER 1 C1 2 OG 2 CB 108.7003.000 MAN-SER 1 O5 1 C1 2 OG 112.3003.000 Engin
Re: [ccp4bb] Dealnig with O-linked mannose
Hi Dmitry, COOT does have the MAN-SER linkage record in its monomer lib, but it won't detect the bond for you. It also haven't provided an interface for the user to specify the bond type yet. The COOT procedure you described is perfectly fine for generating a generic covalent bond record for a PDB file. However without specifying the linkage to be MAN-SER you do lose some restraints for the bond angles (as Engin pointed out in his post regarding phenix.refine). It seems that refmac is the only program (that I've used so far) that will automatically detect the N- and O-glycan linkages and put that information into the header of a PDB file. This seems to be the easiest way of generating a proper LINKRMAN-SER line in the PDB header. Note that refmac writes a LINKR record for its own use, not LINK which is the PDB standard. You can simply manually change the LINKR to LINK to turn it into a PDB standard record, but LINKR works fine for newer versions of COOT too. The advantage of taking this path instead of writing a LINK line manually (or modifying the LINK line you get from COOT) is that you can be sure that the MAN-SER is put in the right columns - PDB format is position-sensitive. Alternatively, here is a LINK line that you can modify to fit your sequence and paste to your PDB file. Just make sure you only replace characters, not inserting/deleting any: LINK C1 MAN B1001 OG SER B 912 MAN-SER For phenix.refine, yes, it does not write any linkage information to the header or use those information stored in PDB headers. But you can always paste that LINK line you get from other places to the PDB files generated by phenix.refine. There is no need to redo the building in COOT every time. The LINK line only specifies the bonding between two atoms. So as long as you haven't renumbered the residues or changed the chain IDs, you can transfer the same LINK line among your PDB files of the same protein (i.e. you do not even have to remake the link in COOT for every structure of the same protein: just paste the LINK line and make sure the residue numbers are right). Zhijie -Original Message- From: Dmitry Rodionov Sent: Thursday, November 21, 2013 4:09 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Dealnig with O-linked mannose Thank you all for helpful suggestions. My question was how to properly connect a mannose to a serine and real-space refine the result. My apologies for not being clear enough. Coot can't find MAN-SER or SER-MAN in it's library (Coot 0.7.1, mon_lib 5.41) It does not automatically make the bond between MAN C1 and OG of SER either. Here is the way I finally made the connection followed by refinement it in Coot: 1) Get monomer... MAN 2) real-space refine MAN into reasonable position 3) Delete hydrogens and reducing hydroxyl Bond is not detected 4) Extensions-Modelling ...-make Link (Click 2 atoms) ... Click on C1 of MAN and OG of SER dashed bond appears 5) Sphere refine A whole different issue that was touched in the replies is how this model will be handled by refinement programs. For the sake of a record, I am using Phenix and it seems to not respect the described link without massaging by means of either pdb.edits or apply_link.def. Also, phenix.refine does not produce LINK records in the output PDB, so step 4 might have to be repeated. Best regards, Dmitry
Re: [ccp4bb] Dealnig with O-linked mannose
It seem the LINK line I provided eariler was chopped by the email system. Here it is again: LINKRC1**MAN*C***1*OG**SER*B*912MAN-SER Simply replace each * with a space and change the residue IDs. Also to clarify the procedure of using refmac to generate the MAN-SER linkage line: 1 In COOT, get monomer-MAN, delete hydrogens, delete O1. Drag the MAN to the proper position. C1 has better be ~1.4A away from OG and mind the anomer conformation. It seems that refmac detects covalent bonds mainly based on the distances. If you accidentaly put the ligand too close to something it should not be linked to (happens when you have poor electron density for positioning the ligand), refmac might complain that a new ligands is found but the geometry information is not provided. In such case the error message is a little misleading to the user: it is a new covalent bond, not a new monoer that's not described. 2 Merge molecules, Save PDB. For linkages that can be found in the standard monomer lib, you do not need to make the bond in COOT. It is refmac that will add this record. 3 Send the saved PDB to refmac. Do restrained refinement. 4 Check the resulting PDB to see if you now have the LINKR...MAN-SER line. And in the refmac log you can find this line: WARNING : link:MAN-SER is found dist = 1.178 ideal_dist= 1.439 ch:BB res: 912 SER at:OG .-CC res: 1 MAN at:C1 . Zhijie -Original Message- From: Dmitry Rodionov Sent: Thursday, November 21, 2013 4:09 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Dealnig with O-linked mannose Thank you all for helpful suggestions. My question was how to properly connect a mannose to a serine and real-space refine the result. My apologies for not being clear enough. Coot can't find MAN-SER or SER-MAN in it's library (Coot 0.7.1, mon_lib 5.41) It does not automatically make the bond between MAN C1 and OG of SER either. Here is the way I finally made the connection followed by refinement it in Coot: 1) Get monomer... MAN 2) real-space refine MAN into reasonable position 3) Delete hydrogens and reducing hydroxyl Bond is not detected 4) Extensions-Modelling ...-make Link (Click 2 atoms) ... Click on C1 of MAN and OG of SER dashed bond appears 5) Sphere refine A whole different issue that was touched in the replies is how this model will be handled by refinement programs. For the sake of a record, I am using Phenix and it seems to not respect the described link without massaging by means of either pdb.edits or apply_link.def. Also, phenix.refine does not produce LINK records in the output PDB, so step 4 might have to be repeated. Best regards, Dmitry
Re: [ccp4bb] Dealnig with O-linked mannose
Hi Dmitry, I think the best way is not to make a new monomer. MAN-SER and MAN-THR linkages do exist in the ccp4 monomer lib. If you simply build a mannose with its O1 removed and put the C1 ~1.4 Angstrom to the OG of the serine I think refmac should be able to detect the linkage. When this happens, you should see a LINKR MAN-SER line in the header of the resulting PDB file. COOT may not show a bond line for linkr record, but the real space refine should work fine. If you need to refine the structure with phenix.refine then you need to make an edit file to specify that the mannose C1 is linked to the ser OG by a covalent bond. Zhijie -Original Message- From: Dmitry Rodionov Sent: Wednesday, November 20, 2013 1:49 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Dealnig with O-linked mannose Good day! I am refining what appears to be O-mannosylated protein structure. In my hands Coot (0.7.1) does not form the SER-MAN bond automatically. I made a SER-MAN.cif with jLigand, which takes care of the glycosidic bond. However, now the peptide bonds are not made to this custom residue (same chain, consecutive numbering). Am I doing something wrong? How can I fix this? Many thanks, Dmitry
