Hi Gustavo,
If I understand you correctly, you are concerned about N-glycans
(N-glycosylation) on your proteins. According to your description, you
have 2 protein molecules in each ASU, each bearing one potential
N-glycan site. Then there are only two N-glycan sites you need to build
for each dataset ( I suppose you are not going to deposit everyone of
them? ). In most cases we do not see much ordered part of the N-glycans,
usually just one or both of the core GlcNac (NAG) residues . If this is
the case then it is not much work.
In very lucky cases you may see one or two rather complete N-glycans. I
think insect cells produce mostly pauci-mannose N-glycans (mostly
Man3-GlcNac2, less frequently Man4-GlcNac2) possibly carrying
core-fucosylation on the innermost GlcNAc. Note that although mammals
only have core 1,6 fucose, insects may also have an additional core 1,3
fucose, discussed and illustrated in the following papers:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3589692/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3647355/
I am not aware of an automated procedure for building complex glycans
such as the N-glycans, so take extreme care making the correct linkages
if you have to build more than the two core NAGs. The Man3Gn2-Asn should
be
Man_alpha1,3-[Man_alpha1,6]-Man_beta1,4-GlcNac_beta1,4-GlcNAc_beta-Asn.
The core fucose(s), if present, will be alpha1,6- or alpha1,3- linked to
the innermost GlcNAc. Note that the core alpha1,3 fucose modification
occurs only when the core alpha1,6 is already there.
Zhijie
On 28/07/2017 1:43 PM, Gustavo Machado Alvares De Lima wrote:
Hello everybody,
I was looking for suggestion on the best way to identify and refine
carbohydrates in a protein. This is the scenario:
- I have 10 molecules in the biological assembly
- I used sf9 expression system, so I have random glycolisation and types of
glycolisations. I have some hint about possible glycolisation sites
- I believe I have only 1 glysolisation per monomer
- I have 2 molecule per AU. Space group C2
I would like (even if I have to write down a script for it) to track this
glycolisation, identify the most probable ones, determine the restrictions for
each and refine them. I could do it by hand on every refinement cycle (or a
couple of cycles), but for many datasets it would take ages.
What protocol would you suggest?
I really appreciate any help in this subject.
Regards,
Gustavo
