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Hello Oliver,

Many thanks for your reply. The disaccharide unit I used to build heparin is,  2-O-sulfo-alpha-L-iduronic acid (LIdopA[2S]a) 1->4 linked to  2-deoxy-2-sulfamido-alpha-D-glucopyranosyl-6-O-sulfate (DGlcpNS[6S]a). The heparin I build have two of this disaccharide unit linked together. Each disaccharide unit have one carboxyl and three sulfate attached ( carboxyl and one sulfate attached to iduronic acid, and two sulfate attached to 2nd and 6th position of glucopyranosyl moiety). Thus carry a total of -8 charge.

The GAG builder generated structure have deprotonated carboxyl group, thus carry a -ve charge on carboxyl group as well.

Please correct me if I am wrong here.

Thanks
Anu

On Wed, Nov 18, 2015 at 12:26 PM, Oliver Grant <[log in to unmask]> wrote:
Hi Anu,

Each of your sulfate and iduronic acid residues carry a net -1 charge, making the total charge on your GAG -6. I need more information on why you want it to be -8 before I can advise you. The sequence you showed above would not have a -8 charge. It could be that the sequence you require has a sulfate at both the 6 and 3 position of the GlcNAc (as occurs in "heparin") e.g. DGlcpNAc[3S,6S]a1-4LIdopA[2S]a1-4DGlcpNAc[3S,6S]a1-4LIdopA[2S]a1-OME
This would have a -8 charge.


Oliver

On Tue, Nov 17, 2015 at 8:03 PM, Oliver Grant <[log in to unmask]> wrote:

LINK cards can be ignored. 


Yes that is what TER cards will do in tleap.


Tleap was designed for proteins and thus assumes a linear sequence of residues. It will bond the tail atom of a residue to the head atom of the next residue unless you place a TER card between them. Your molecule is technically branched as you have sulfate residues attached. Best is to add TER cards between EVERY residue and explicitly bond them with tleap bond commands. If you separate the LINK cards into a separate file, you may run the attached LINK2bond on it to produce tleap bond commands. Just chmod +x LINK2bond once you download it.
We also find all this overly complex! It will soon be a thing of the past.

On 17 Nov 2015 5:27 p.m., "anu chandra" <[log in to unmask]> wrote:
Thanks to all for your helpful reply.

As a beginner, I build the heparin unit - LIdopA[2S]a1-4DGlcpNS[6S]a1-4LIdopA[2S]a1-4DGlcpNS[6S]a1-OH - using GAG builder in order get familiarize with the Glycam naming, before renaming the heparin pdb file I have.

Here, I am little confused with the format of the generated pdb file. I have noticed that top of the pdb file have  following details mentioned,

LINK         O1  ROH     1                 C1  UYS     2
LINK         O6  UYS     2                 S1  SO3     9
LINK         O4  UYS     2                 C1  YuA     3
LINK         O4  YuA     3                 C1  UYS     4
LINK         O2  YuA     3                 S1  SO3     8
LINK         O6  UYS     4                 S1  SO3     7
LINK         O4  UYS     4                 C1  2uA     5
LINK         O2  2uA     5                 S1  SO3     6

Is this just for the information or do I need to keep this information in my pdb file as well?


I have also noticed that there is a TER card in between the monosaccharide units in amber formatted pdb file (1_AMBER.pdb). For e.g.,

HETATM   25  O5  UYS     2       8.455   8.107  -2.556  1.00  0.00           O
HETATM   26  O4  UYS     2       8.472   7.396  -6.246  1.00  0.00           O
TER
HETATM   27  C1  YuA     3       9.694   7.499  -7.062  1.00  0.00           C
HETATM   28  H1  YuA     3      10.522   7.063  -6.507  1.00  0.00           H

If I am right, usually  TER card is placed only when a residue get terminated ( i.e not bonded to the next residue). May I know why TER card is inserted in between the covalently linked monosccharide units?


Also, how do I re-adjust the atomic charges if I need to  set the total charge on heparin to 8 -ve?


Many thanks once again
Anu


On Tue, Nov 17, 2015 at 2:12 PM, Oliver Grant <[log in to unmask]> wrote:
Hi Anu,

No you won't have to do that. When connecting sulfates you just adjust the charge on the connecting oxygen/nitrogen in tleap. E.g:
set mol.160.O6 charge -0.437
I adjusted it by + 0.031. Replace mol.160 as appropriate for your system.
For attaching a sulfate to the N of a b-GlcN you need to adjust the charge on the linking N by -0.057. You can find out what charge the N is in tleap with e.g:
charge mol.3.N2





Oliver

On Tue, Nov 17, 2015 at 2:28 PM, anu chandra <[log in to unmask]> wrote:
Hi Oliver,


Thanks for your prompt reply. A quick query here is about the RESP charge calculation. I have noticed that in Glycam, the atomic partial charges are derived for monosaccharide units and also mentioned about change in charge while adding functional groups like sulfates. Do I need to do any  geometry optimization and partial charge calculation for the heparin I am using?. The heparin contains two disaccharide units.


Many thanks

Anu

On Mon, Nov 16, 2015 at 6:40 PM, Oliver Grant <[log in to unmask]> wrote:
Hi Anu,

I forgot about adding sulfates. You'll also have to follow this page:

Oliver

On Mon, Nov 16, 2015 at 6:12 PM, Oliver Grant <[log in to unmask]> wrote:
Hi Anu,

I think this is the standard protocol right now:

Add these lines to your tleap input file. Insert your own paths and forcefields that you are using.

source /programs/amber/dat/leap/cmd/leaprc.GLYCAM_06j-1
source /programs/amber/dat/leap/cmd/leaprc.ff14SB

You can see I'm using AMBER14. Now the hard part is getting your ligand named correctly so that the correct GLYCAM parameters get assigned when you load it into tleap. Protein residues have standard names in the PDB, but unfortunately carbs do not. I assume you have a PDB format file of the co-complex. You have to manually edit the PDB file with a text editor, renaming the residues and atom names to GLYCAM nomenclature. See here for the nomenclature to use: http://glycam.org/docs/forcefield/glycam-naming-2/
If this is your first time, it will be useful for you to build the sugar on our website, download the PDB file, and open it up in a text editor. You will see what format the carbohydrate residues and atom names need to be in. Arunima Singh and others from the group have made a GAG builder for this: http://glycam.org/tools/molecular-dynamics/oligosaccharide-builder/build-glycan?id=8. Write back if you have any trouble with it. User feedback is precious to us.

In the very near future (we are testing the software right now) we'll be releasing software that renames everything for you, so this will be so much easier.

All the best,

Oliver

On Mon, Nov 16, 2015 at 3:35 PM, SUBSCRIBE GLYCAM-L Anu <[log in to unmask]> wrote:
Hello Glycam users,

I am planning to do MD simulation of a protein-ligand complex in which ligand is heparin, made of 2-O-sulfo-aplha-L-iduronic acid and 2-deoxy-2-sulamido-alpha-D-glucopyranosyl-6-O-sulfate. I am using Amber 12 MD package for simulation. It will be of great help if somebody can help me to get the Amber compatible Glycam  parameters for heparin.


Many thanks
Anu