Dear sugar loving people,
I have been working with a lot of glycoproteins (up to 30 %
carbohydrates) at resolutions as "bad" as 2.8 Å. Nevertheless, I was
able to built sometimes about 10 sugar moieties/carbohydrate chain.
Although, sugar molecules usually have a somewhat bulky density, and
don´t have such distinct structural features like in proteins (e.g.
main chain, side chain, C=O bump) one can use geometric restraints to
place the sugars correctly with a high probability. I other words I
usually first superimpose the e.g. O1 of the sugar molecule with the N
of the Asn or O of the Ser/Thr or with the e.g. O4 of the previous
sugar (looking from Asn/Ser/Thr). Then I just rotate about the
glycosidic bond or only about the O1 (e.g. C1-O4 for a beta1-4) and
try to fit the density as good as possible. Often real space
refinement in COOT helps after this stage to optimize. Then I check
the interactions of the newly placed sugar with its close
neighbourhood. Well defined sugars, and only those you can see in a
crystal structure, make practically with each of their OH groups or
any other polar atom/group (e.g. the N in NAG) at least one H-bond,
either with the protein, with other neighbouring sugars, or with
waters or even with other solvent molecules like SO4. Then you should
also check reasonable van der Waals distances of non-polar atoms. If
you find a conformation where you have the optimum number of H-bonds
and no outliers (e. g. < 3.2 Å) of close non-bonding contacts, you
most likely have placed your sugar molecule correctly.
In e.g. REFMAC with the review mode you can then check if you have a
alpha or beta glycosidic bond and if this is what you expect.
BR,
Klaus
Am 22.04.2010 um 19:13 schrieb tirumal:
Thanks to all who responded. 180 degrees flip of the problematic
NAGs, did help.
> At the moment, there is no substitute for knowledge when building
carbohydrates - it >would be a substantial improvement I think if
someone added intelligent carbohydrate >validation tools into Coot.
If you have a poor density (which I guess, generally is the case for
large glycoprotein structures) you have to depend on trial and error
strategy to get the right NAG conformation. I don't know how other
refinement programs handle this, but after Phenix.refinement run,
one has to definitely check the geometry of the NAGs carefully.
Hope to see a validation tool for NAGs in Coot soon.
Tirumal
--- On Wed, 21/4/10, Garib Murshudov <[email protected]> wrote:
From: Garib Murshudov <[email protected]>
Subject: Re: [ccp4bb] geometry problems with sugars
To: [email protected]
Date: Wednesday, 21 April, 2010, 9:58
JED's example is very illustrative and it shows that chirality may
need to be added to this link definition. then sugar validation may
be easier (at least ASN-NAG with only one sugar). If chirality is
wrong then rotate around ND2-C1bond as a rigid group. Just like you
do with rotamers. Here you have only two orientations.
Garib
On 21 Apr 2010, at 14:20, Paul Emsley wrote:
> Garib Murshudov wrote:
>> As I see there is no chirality definition for NAG-ASN link
(perhaps there should be but then people will be unhappy even more).
>> Only reason i can see for this flattening is conflict between
geometry and electron density. Your example shows that even if
electron density is weak it may play a role and correct orientation
of sugar may matter.
>>
>
> I agree, and with JED too. More tests suggest that if I put the
NAG into the density the wrong way round, Coot will happily flatten
the C1. So, my guess would be that if you rotated your NAG 180
degrees round a vector ~ NG--(midpoint of C3,C4) and re-refined,
then things would improve.
>
> At the moment, there is no substitute for knowledge when building
carbohydrates - it would be a substantial improvement I think if
someone added intelligent carbohydrate validation tools into Coot.
>
> Paul.
Dr. Klaus Piontek
Albert-Ludwigs-University Freiburg
Institute of Organic Chemistry and Biochemistry, Room 401 H
Albertstrasse 21
D-79104 Freiburg Germany
Phone: ++49-761-203-6036
Fax: ++49-761-203-8714
Email: [email protected]
Web: http://www.chemie.uni-freiburg.de/orgbio/w3platt/
