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Hi Dan, the best way is to built your sugar tree from the monomers subsequently. Starting from GlcNAC. Remove the atoms to link it to the Asn. This link is surely defined in the mon_lib_list. You find there the information which atoms have to be removed. Than proceed to load the next monomer (NAG again) Look for the respective link definition in the mentioned list and remove the respective atoms and so on. That is easily don in COOT. That should give you the respective sugar tree without the need of any special library. You might switch on the Torsion restraints in Coot and Refmac to get a proper geometry. I recommend to check your sugars with privateer after your refinement, because often sugars get distorted due to their inherent flexibility which results often in fuzzy density. If you need more help, let me know. Cheers Christian Am 27.07.2015 um 18:28 schrieb Dan Freed: > Hi all, > > I'm refining a protein in Refmac with several N-glycosylation sites. Refmac was able to recognize and successfully write a .lib parameter file for sites that had density for the first three sugars (NAG-NAG-BMA), and I could use this file in Coot and Refmac without any issues. However, I am having trouble making a .cif or .lib file for glycosylation sites that have density for the next two sugars (1,3- and 1,6-alpha glycosidic MAN-BMA linkages, forming the core pentasaccharide Man(3)GlcNAc(2)). > > I've numbered the residues consecutively (no overlap with protein residues), designated all of the sugar atoms as HETATM, and specified LINK lines in the pdb file. However, Refmac still fails, and won't write a .lib file that I can use in subsequent runs. I haven't had luck with .cif files generated from PRODRG, can't find a file in HIC-Up or Ligand Expo, and I'm not entirely sure how to run eLBOW on more than one 'ligand', since I technically have a NAG, BMA and MAN oligosaccharide. > > I'd be very grateful if someone could explain how to make (or find) a .cif file for N-linked Man(3)GlcNAc(2). Thanks in advance! > > Best, > Dan >