Re: [COOT] Conflicting opinions on what constitutes an alpha1-6 L-fucose between Coot and Privateer

Paul Emsley Tue, 23 Apr 2024 18:04:11 -0700

We are talking about different things, I think.

Coot's glyco builder builds alpha 1-6 linked FUC correctly.

Coot's glyco validator used to (until this evening) identify the alpha1-6 linked FUC as a beta anomer (it no longer does).


See for example the G8 FUC in 8q5u: Coot 0.9.8.94 now prints:

ASN [spec: 1 "A" 297 ""]
                                    pyr-ASN NAG [spec: 1 "G" 1 ""]
                                BETA1-4 NAG [spec: 1 "G" 2 ""]
                            BETA1-4 BMA [spec: 1 "G" 3 ""]
                        ALPHA1-3 MAN [spec: 1 "G" 4 ""]
                    BETA1-2 NAG [spec: 1 "G" 5 ""]
                        BETA1-6 MAN [spec: 1 "G" 6 ""]
                    BETA1-2 NAG [spec: 1 "G" 7 ""]
                                ALPHA1-6 FUC [spec: 1 "G" 8 ""]

Paul.


On 24/04/2024 01:38, Engin Özkan wrote:

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Hi Markus,

I am using Coot that comes with CCP4 8, which has FUC (alpha-L-Fucose)with an oxygen on C1 in the axial position. Which is the correct form,I believe.

Using Coot 0.9.8.93, I get my alpha1-6 and alpha1-3 (insect) Fucosesinserted correctly. I had trouble understanding your description: Youmean you get Coot to insert a FUC with the glycosidic bond goingequatorial, instead of axial, to Fucose?


This is how it works for me:

https://www.dropbox.com/scl/fi/yjbzstojjivmfpqdlz2os/Screen-Recording-2024-04-23-at-7.32.14-PM.mov?rlkey=zvvk7apd19wk61kt21qyc42l5&dl=0

I'll see what happens when I update to the new Coot version where Paulhas switched the logic.

By the way, privateer tells me I have zero errors and the correctalpha anomer for Fucose.


Fuc913-a6-GlcNAc911-b-ASN258
WURCS=2.0/2,2,1/[a2122h-1b_1-5_2*NCC/3=O][a1221m-1a_1-5]/1-2/a6-b1
...

PDB Sugar Q Phi Theta Detected type Cnf <Bfac> Ctx Ok?---- ------------ ----- ------ ------ ------------------- --- ------ --- -----test NAG-A-911 0.570 280.48 5.29 beta-D-aldopyranose 4c1 47.88 (n) yestest FUC-A-913 0.535 92.53 177.32 alpha-L-aldopyranose 1c4 70.76 (n) yes

...
Wrong anomer: 0

Best,


Engin


On 4/18/24 4:35 PM, Markus Meier wrote:

Hi Engin,

hmm, it appears I was using the monomer library fromhttps://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/dependencies/monomers-2023-01-02-23:57:29.tar.gz(it gets installed automatically with Coot's build-it script).

However, the files in question (FUC.cif, NAG.cif, BMA.cif andMAN.cif) are identical to current CCP4 8.0.018 version of the library(installed by CCP4 Update Manager) as well as version ccp4-8.0.018and version ccp4-8.0.016 on GitHub(https://github.com/MonomerLibrary/monomers)


MD5 checksums on these files are
9cfba4ac24f056d648df8227316f788b  monomers/f/FUC.cif
958ac5b19a92ceec604d233457944b2a  monomers/n/NAG.cif
697dd5242334dd99dad9ce43133c6382  monomers/b/BMA.cif
9d68ff54296ef2f4622005efb4eb080b  monomers/m/MAN.cif

If they are different on your machine, please let me know.

I will switch monomer libraries anyway and do some testing.

I also was switching back and forth between my locally compiled coot(0.9.8.92) and the the CCP4 distributed version (0.9.8.93 EL). I willcheck if there is a difference between the two.


Thank you very much for your suggestion!

With best regards,
Markus

On 2024-04-18 13:48, Engin Özkan wrote:

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Hi Markus,

There were reports to the ccp4bb and phenixbb back in 2011 about
something similar. The monomer library files for FUC and/or the linkage
definitions for refmac/phenix were not right. I think. My recollection
is that this was corrected, and I could not reproduce your observation.

I also had private discussions with Garib Murshudov, who werecorrecting

the glycan monomer problems back in ~2011.

I could find old links to some of the discussions on bulletin board
archives:

https://phenix-online.org/pipermail/phenixbb/2011-November/017958.html

https://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg13968.html

I am curious if something got reverted or if you have some ancient
library definitions somewhere. But I hope this gives some ideas.

Best,

Engin


On 4/17/24 3:07 PM, Markus Meier wrote:

Hi Paul,

thank you for implementing the Glyco module in Coot!

I used its "Add N-linked Glycan" function to build an N-linked glycan
which contains L-fucose with an alpha1-6 glycosidic linkage on the
first N-acetyl glucosamine of the tree.

However, after exporting the tree, I checked it with privateer and
unfortunately privateer flags the L-fucose as having the wrong
anomeric linkage - beta 1-6 :-(
Coot reports the same as being in the alpha 1-6 anomeric form.

I then manually built the opposite anomeric form which privateer

happily accepts as being alpha1-6. However, Coot reports it as beta1-6.


In my understanding, the alpha configuration of a glycosidic linkage
should have the oxygen at the anomeric centre and the -CH3 group at
opposite faces of the sugar ring, so I have to agree with privateer on
this one.

Please check you code.


I have attached the two glycan trees and here is the output of
privateer and Coot for both. Coot and privateer versions are given at
the bottom.

Btw. Neither privateer nor refmac5 can read the mmCIF files that Coot
produces with the "Extract this Tree" functions. All the
_atom_site.label_*_id entries are empty!

N133_tree_privateer_alpha_anomer_FUC.cif:

  Privateer:
    Detailed validation data
    ------------------------

    PDB       Sugar       Q     Phi    Theta      Detected type
Cnf  <Bfac>  Ctx   Ok?
    ----  ------------  -----  ------  ------ -------------------
---  ------  ---  -----
    N133  NAG-A-459     0.597   63.27    2.85 beta-D-aldopyranose
4c1  28.09   (n)  yes
    N133  NAG-A-460     0.603  145.39    2.15 beta-D-aldopyranose
4c1  36.34   (n)  yes
    N133  BMA-A-461     0.550  333.80    4.39 beta-D-aldopyranose
4c1  45.25   (n)  yes
    N133  MAN-A-462     0.628    6.26    5.03 alpha-D-aldopyranose
4c1  46.22   (n)  yes
    N133  MAN-A-463     0.628    4.87    5.87 alpha-D-aldopyranose
4c1  50.37   (n)  yes
    N133  FUC-A-464     0.615  162.25  174.51 alpha-L-aldopyranose
1c4  31.24   (n)  yes <-

    Partially occupied monosaccharides, if any, are marked with an
asterisk (*)

    SUMMARY:

       Wrong anomer: 0
       Wrong configuration: 0
       Unphysical puckering amplitude: 0
       In higher-energy conformations: 0

       Privateer has identified 0 issues, with 0 of 6 sugars affected.
    Coot output: