# Pdb2gmx does not make disulfide bond between peptide chains **URL:** \ **Category:** User discussions\ **Tags:** pdb2gmx\ **Created:** [May 3, 2023, 1:15am UTC](https://gromacs.bioexcel.eu/t/pdb2gmx-does-not-make-disulfide-bond-between-peptide-chains/6325 "2023-05-03T01:15:18Z")\ **Posts on this page:** 6\ **Page:** 1
**Author:** ![TGaziri](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/tgaziri/32/5191_2.png) [@TGaziri](https://gromacs.bioexcel.eu/u/TGaziri)\ **Post date:** [May 3, 2023, 1:15am UTC](https://gromacs.bioexcel.eu/t/pdb2gmx-does-not-make-disulfide-bond-between-peptide-chains/6325/1 "2023-05-03T01:15:18Z")
Gromacs version: 2021.5 Modification: No Force Field: Charmm C36M Hello, I’m trying to generate the topology for a peptide with 2 identical chains linked by a disulfide bond at the n-terminus. But I can’t get `pdb2gmx ` to create the disulfide bond. `pdb2gmx` creates the topology without the disulfide bond. I even tried to modify the `specbond.dat` file to allow disulfide bonds for longer distances but it doesn’t work. I’ve tried the following commands: `gmx pdb2gmx -f dimer.pdb -o dimer_ss.gro -ignh -ss` `gmx pdb2gmx -f dimer.pdb -o dimer_ss.gro -ignh -ss -merge all ` `gmx pdb2gmx -f dimer.pdb -o dimer_ss.gro -ignh -ss -merge inter` `gmx pdb2gmx -f dimer.pdb -o dimer_ss.gro -ignh -ss -merge all -chainsep ter` None of them worked. My files can be accessed in the following link: [Google Drive](https://drive.google.com/drive/folders/1U7VES0lWqWjpjb4Ve-EvWhFDL44VWaL3?usp=sharing) Any help will be appreciated. ---
**Author:** ![jalemkul](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/jalemkul/32/18_2.png) [@jalemkul](https://gromacs.bioexcel.eu/u/jalemkul)\ **Post date:** [May 3, 2023, 1:42pm UTC](https://gromacs.bioexcel.eu/t/pdb2gmx-does-not-make-disulfide-bond-between-peptide-chains/6325/2 "2023-05-03T13:42:07Z")
> [@TGaziri](#): > > gmx pdb2gmx -f dimer.pdb -o dimer\_ss.gro -ignh -ss -merge all This is the correct approach, but the file you have provided in the Google Drive link called “dimer.pdb” has two identical chains (with identical coordinates) separated by an END card, so it seems to me only one chain would ever be read. But you can’t have two chains with the same coordinates anyway, so something is simply wrong with the coordinate file. It is essential when diagnosing `pdb2gmx` issues to provide the complete, copied and pasted, terminal output. There is a ton of useful information there that can be used for troubleshooting. ---
**Author:** ![TGaziri](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/tgaziri/32/5191_2.png) [@TGaziri](https://gromacs.bioexcel.eu/u/TGaziri)\ **Post date:** [May 3, 2023, 8:53pm UTC](https://gromacs.bioexcel.eu/t/pdb2gmx-does-not-make-disulfide-bond-between-peptide-chains/6325/4 "2023-05-03T20:53:56Z")
Good evening, thank you for your reply professor. I’ve updated the files in the link to include the fulll pdb2gmx output `pdb2gmx_output.txt`. Also I’ve tried creating a new structure to see if it would work, but it didn’t. It’s odd that pdb2gmx has written the topology for both chains but no S-S bond. Also it’s weird that visualizing with pymol, showed 2 chains with different postions. Except for the forcefield options that I couldn’t fit in here the output was: ```auto Back Off! I just backed up topol.top to ./#topol.top.1# Processing chain 1 (92 atoms, 14 residues) Analysing hydrogen-bonding network for automated assignment of histidine protonation. 18 donors and 14 acceptors were found. There are 14 hydrogen bonds Will use HISE for residue 6 Will use HISE for residue 6 Identified residue CYS2 as a starting terminus. Identified residue LEU8 as a ending terminus. Identified residue CYS2 as a starting terminus. Identified residue LEU8 as a ending terminus. 8 out of 8 lines of specbond.dat converted successfully Special Atom Distance matrix: CYS2 HIS6 CYS2 SG6 NE230 SG52 HIS6 NE230 0.529 CYS2 SG52 0.093 0.470 HIS6 NE276 0.487 0.943 0.529 Start terminus CYS-2: NH3+ End terminus LEU-8: COO- Start terminus CYS-2: NH3+ End terminus LEU-8: COO- Opening force field file ./charmm36-jul2022-napht.ff/aminoacids.arn Checking for duplicate atoms.... Generating any missing hydrogen atoms and/or adding termini. Now there are 14 residues with 192 atoms Making bonds... Number of bonds was 192, now 192 Generating angles, dihedrals and pairs... Before cleaning: 478 pairs Before cleaning: 488 dihedrals Keeping all generated dihedrals Making cmap torsions... There are 10 cmap torsion pairs There are 488 dihedrals, 38 impropers, 346 angles 478 pairs, 192 bonds and 0 virtual sites Total mass 1367.658 a.m.u. Total charge 0.000 e Writing topology Back Off! I just backed up posre.itp to ./#posre.itp.1# Writing coordinate file... Back Off! I just backed up dimer_ss.gro to ./#dimer_ss.gro.1# --------- PLEASE NOTE ------------ You have successfully generated a topology from: dimer_new.pdb. The Charmm36-jul2022-napht force field and the tip3p water model are used. --------- ETON ESAELP ------------ GROMACS reminds you: "The Feeling of Power was Intoxicating, Magic" (Frida Hyvonen) ``` Although it has sucessfully written the topology it does not contain the ss bond ---
**Author:** ![jalemkul](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/jalemkul/32/18_2.png) [@jalemkul](https://gromacs.bioexcel.eu/u/jalemkul)\ **Post date:** [May 4, 2023, 2:05am UTC](https://gromacs.bioexcel.eu/t/pdb2gmx-does-not-make-disulfide-bond-between-peptide-chains/6325/5 "2023-05-04T02:05:36Z")
> [@TGaziri](#): > > Although it has sucessfully written the topology it does not contain the ss bond The two SG atoms are too close to one another. Their distance in the coordinate file is 0.093 nm, according to the screen output. The typical S-S length is on the order of 0.2 nm, which is what `specbond.dat` uses. The tolerance for this value is ±10%, so the distance needs to be within 0.18 - 0.22 nm for it to write the bond. ---
**Author:** ![TGaziri](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/tgaziri/32/5191_2.png) [@TGaziri](https://gromacs.bioexcel.eu/u/TGaziri)\ **Post date:** [May 4, 2023, 9:21pm UTC](https://gromacs.bioexcel.eu/t/pdb2gmx-does-not-make-disulfide-bond-between-peptide-chains/6325/6 "2023-05-04T21:21:19Z")
Thank you very much for your replay professor, it finally worked. ---
**Author:** ![shivam1](https://avatars.discourse-cdn.com/v4/letter/s/b3f665/32.png) [@shivam1](https://gromacs.bioexcel.eu/u/shivam1)\ **Post date:** [July 31, 2023, 6:57am UTC](https://gromacs.bioexcel.eu/t/pdb2gmx-does-not-make-disulfide-bond-between-peptide-chains/6325/7 "2023-07-31T06:57:21Z")
Hello @jalemkul Is there any entry of S-S bonds in topology file? If it is, under which section/header can I confirm? I have created topology with and without `-ss` flag. Both gave me same results. Thanks