Dear Dan,
the devil lies probably in the detail. You can do it in phenix as well,
but it will be similar easy/complicated. You could send me the
coordinates of the Asn plus the sugars and I have a look what goes
actually wrong. There is no need for anything else.
Christian
Am 27.07.2015 um 20:33 schrieb Dan Freed:
> Thanks for your quick replies, Christian and Engin. I'm not sure if I explained my problem very well.
>
> The problem is not with building the sugar chain, it is with refinement in Refmac. I have built the pentasaccharide successfully, and I have also specified the proper "LINK" and "LINKR" designations in the pdb file. Coot has no problem recognizing these linkages, and performing real space refinement of the pentasaccharide. However, Refmac cannot handle it and fails every time. The .cif file that Refmac makes by default does not work in subsequent runs, either.
>
> Maybe I should just switch to Phenix for the remainder of the refinement process...
>
>
>
> ----- Original Message -----
> From: "Engin Özkan" <[log in to unmask]>
> To: "Dan Freed" <[log in to unmask]>, [log in to unmask]
> Sent: Monday, July 27, 2015 2:33:29 PM
> Subject: Re: [ccp4bb] N-linked Man3GlcNAc2 cif file
>
> I am not that familiar with Refmac, but I don't think you should be
> defining sugar chains as one cif. Refmac uses the standard monomer
> library (as Phenix did), which knows the sugars and the types of links
> contained in N-linked sugars (and other ones, too). I checked the Refmac
> manual, and the interesting keywords there appear to be LINK and SUGAR.
>
> The list of links known to Refmac are here:
>
> http://www.ccp4.ac.uk/html/refmac5/dictionary/list-of-ligands.html#links
>
> Among those, "NAG-ASN" would define the first Asparagine to N-acetyl
> glucosamine. "BETA1-4" would link NAG#1 to NAG#2, "BETA1-4" would link
> NAG#2 to BMA, "ALPHA1-3" and "ALPHA1-6" would link the BMA to the MANs, etc.
>
> Engin
>
> On 7/27/15 12:28 PM, Dan Freed wrote:
>> 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
>
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