# How to prepare buffer salt for Gromacs?

**URL:** <https://gromacs.bioexcel.eu/t/how-to-prepare-buffer-salt-for-gromacs/2076>\
**Category:** User discussions\
**Tags:** simulation-setup\
**Created:** [May 1, 2021, 10:06am UTC](https://gromacs.bioexcel.eu/t/how-to-prepare-buffer-salt-for-gromacs/2076 "2021-05-01T10:06:31Z")\
**Posts on this page:** 5\
**Page:** 1

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**Author:** ![lanselibai](https://avatars.discourse-cdn.com/v4/letter/l/e9c0ed/32.png) [@lanselibai](https://gromacs.bioexcel.eu/u/lanselibai)\
**Post date:** [May 1, 2021, 10:06am UTC](https://gromacs.bioexcel.eu/t/how-to-prepare-buffer-salt-for-gromacs/2076/1 "2021-05-01T10:06:31Z")

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GROMACS version:2020  
GROMACS modification: No  
Here post your question

Hi, I plan to use “insert-molecules” to add HEPES buffer into the simulation. [Justin’s tutorial](http://www.mdtutorials.com/gmx/biphasic/01_genconf.html) uses `PRODRG` to generate coordinate` gro` file and topology `top` file. I think I should firstly obtain a PDB of HEPES, where to get it?

Also, I need the HEPES in acid form (protonated) and basic form (deprotonated), how to get them?

In addition, at which stage should I insert the HEPES, is it after `pdb2gmx`, `editconf`, `solvate`, or adding ions?

Is there a prefered force-field to handle the buffer salt? Now I use amber14sb, is that okay?

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**Author:** ![alevilla](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/alevilla/32/439_2.png) [@alevilla](https://gromacs.bioexcel.eu/u/alevilla)\
**Post date:** [May 4, 2021, 9:15am UTC](https://gromacs.bioexcel.eu/t/how-to-prepare-buffer-salt-for-gromacs/2076/2 "2021-05-04T09:15:43Z")

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Hi,

> [@lanselibai](#):
>
> I should firstly obtain a PDB of HEPES, where to get it?

You can take pdb for HEPES from a pdb databank (alone or in complex with a biomolecule for example) And/Or you can build the molecule using a visualization software (some visualization softwares have the option to build molecules or fragment of molecules) and save as pdb.

> [@lanselibai](#):
>
> Is there a prefered force-field to handle the buffer salt? Now I use amber14sb, is that okay?

choose force field parameters that are in line with the force field you used to describe rest of the system (in your case amber14sb, as far as I understood)

> [@lanselibai](#):
>
> In addition, at which stage should I insert the HEPES, is it after `pdb2gmx` , `editconf` , `solvate` , or adding ions?

I assume you want to simulate a biomolecules in HEPES and you have the coordinate file and topology file (itp file) for HEPES in the appropriate protonation form. I suggest the following step

- pre-processing of the biomolecule (pdb2gmx)
- box definition and solvation of biomolecule (gmx editconf gmx solvate)
- insert HEPES molecules (and include the corresponding itp file in the topol.top.
- neutralize the system

Best regards  
Alessandra

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<div class="post-metadata">

**Author:** ![lanselibai](https://avatars.discourse-cdn.com/v4/letter/l/e9c0ed/32.png) [@lanselibai](https://gromacs.bioexcel.eu/u/lanselibai)\
**Post date:** [May 4, 2021, 11:06am UTC](https://gromacs.bioexcel.eu/t/how-to-prepare-buffer-salt-for-gromacs/2076/3 "2021-05-04T11:06:25Z")

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Thank you very much Alessandra!

Should I` insert-molecules` for the acid form (protonated, 0 charge) and basic form (deprotonated, -1 charge) of the HEPES separately? How can I prepare the deprotonated HEPES?

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<div class="post-metadata">

**Author:** ![alevilla](https://dub1.discourse-cdn.com/flex017/user_avatar/gromacs.bioexcel.eu/alevilla/32/439_2.png) [@alevilla](https://gromacs.bioexcel.eu/u/alevilla)\
**Post date:** [May 5, 2021, 8:13am UTC](https://gromacs.bioexcel.eu/t/how-to-prepare-buffer-salt-for-gromacs/2076/4 "2021-05-05T08:13:07Z")

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Hi,  
Yes, you can insert one type of molecule at the time.  
You can manually build the deprotonated form, or use a graphical software that allows to delete atoms  
Best regards  
Alessandra

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<div class="post-metadata">

**Author:** ![lanselibai](https://avatars.discourse-cdn.com/v4/letter/l/e9c0ed/32.png) [@lanselibai](https://gromacs.bioexcel.eu/u/lanselibai)\
**Post date:** [May 5, 2021, 2:26pm UTC](https://gromacs.bioexcel.eu/t/how-to-prepare-buffer-salt-for-gromacs/2076/5 "2021-05-05T14:26:24Z")

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Thank you, I got that. It would be better if `insert-molecules` can modify the protonation status.