Dear all,

I am trying to refine N-linked carbohydrates with REFMAC5 (CCP4 version
6.0.2) - however - it seems as if I am unable to get the stereochemistry
right (when I tried to deposit the structure to PDB - they complaied about
it). My protein comes from D. melanogaster and I can see density for the
Asn-linked NAG (with a core alpha-1,6-fucose), the second NAG, and three
mannoses. This is the header I have been using (I am not a 100% convinced
it is correct - maybe someone could verify this please?):

LINK C1 NAG A 501 ND2 ASN A 105
NAG-ASN
LINK O4 NAG A 501 C1 NAG A 502
BETA1-4
LINK O4 NAG A 502 C1 BMA A 503
BETA1-4
LINK O6 BMA A 503 C1 MAN A 504
ALPHA1-6
LINK O3 BMA A 503 C1 MAN A 505
ALPHA1-3
LINK O6 NAG A 501 C1 FUC A 506
ALPHA1-6
MODRES NAG A 501 NAG-b-D
RENAME
MODRES NAG A 502 NAG-b-D
RENAME
MODRES BMA A 503 MAN-b-D
RENAME
MODRES MAN A 504 MAN-a-D
RENAME
MODRES MAN A 505 MAN-a-D
RENAME
MODRES FUC A 506 FUC-a-L
RENAME

(1) I'd really appreciate if someone could confirm that I am using the
correct links (i.e. the correct sugars (the protein is exressed in fly
cells) and how they are linked).

(2) Am I using the correct header for handling the sugars - and if not -
what should I include/use instead?

(3) Does someone have a working cif-file which includes all the necessary
restraints for those sugars including the links, which I could use as a
library for REFMAC? PLEASE!!!!

(4) I am a bit confused about the naming convention of mannoses:
MAN-b-D (according HICUP) has the residue code BMA, whereas MAN-a-D has
MAN. However, it seems that in the REFMAC dictionary MAN seems to be used
for both and there does not seem to be a MAN-a-D.cif file either. Can
anyone please comment on this?


THANK YOU SOOO MUCH, Petra


--
Petra Verdino, PhD
Research Associate
The Scripps Research Institute, BCC206
10550 North Torrey Pines Road
CA 92037 La Jolla, USA
tel:1-858-784-2294
fax:1-858-784-2980