# HADDOCK2.4 refinement web portal
**URL:** https://ask.bioexcel.eu/t/haddock2-4-refinement-web-portal/4467
**Category:** HADDOCK
**Created:** [August 26, 2023, 9:07am UTC](https://ask.bioexcel.eu/t/haddock2-4-refinement-web-portal/4467 "2023-08-26T09:07:42Z")
**Posts on this page:** 2
**Page:** 1
### Author:  [@marianvincenzi](https://ask.bioexcel.eu/u/marianvincenzi)
#### Post date: [August 26, 2023, 9:07am UTC](https://ask.bioexcel.eu/t/haddock2-4-refinement-web-portal/4467/1 "2023-08-26T09:07:42Z")
Dear All,
I used Haddock Refinement Interface 2.4 to introduce different puntual mutations in a complex structures. The structures used as input to introduce mutations was a NMR structure of a protein complex with four Histidines not charged and with proton located on ND1 atom for all histidines. The problem was in the output, since 3 out 4 histidines moved the protons to the NE2 atom.
Now I am trying to overcome the issue by using the guru interface and by manually selecting the protonation state and type of histidine (HISD or HISE).
So my question, If I would to reproduce all parameters of Haddock Refinement interface 2.4 but setting manually the protonation state of histidines, How I can do ?
In the first attempt I used the following parameters found in a previous discussion on bioexcel:
In **“Distance Restraints”** :
“_Define center of mass restraints to enforce contact between the molecules_ ” \> True
“_Define surface contact restraints to enforce contact between the molecules_ ” \> True
In **“Sampling parameters”** :
“_Number of structures for rigid body docking (it0)_ ” \> Same as it1 and water, in your case, if you want 10 models per complex I’d suggest to use 200
“_Sample 180 degrees rotated solutions during rigid body EM_ ” \> False
In **“Advanced Sampling Parameters”** :
“_Perform cross-docking_ ” \> False
“_Multiply the number of calculated structures by all combinations_ ” \> True
“_Randomize starting orientations_ ” \> False
“_Perform initial rigid body minimisation_ ” \> False
“_Allow translation in rigid body minimisation_ ” \> False
“_number of MD steps …_ ”\*4 \> 0 for the 4 values (respectively 500/500/1000/1000 by default)
The second questions is concerning “_Multiply the number of calculated structures by all combinations_:
Since the input structures are complexes, it means it leads to combine the chain A of the model 1 of the ensemble with chain B of all models?
Thank you in advance,
Marian
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### Author:  [@amjjbonvin](https://ask.bioexcel.eu/u/amjjbonvin)
#### Post date: [September 17, 2023, 11:31am UTC](https://ask.bioexcel.eu/t/haddock2-4-refinement-web-portal/4467/2 "2023-09-17T11:31:02Z")
The manual settings for refinement are described here:
[https://www.bonvinlab.org/software/haddock2.4/tips/advanced\_refinement/](https://www.bonvinlab.org/software/haddock2.4/tips/advanced_refinement/)
> The second questions is concerning “_Multiply the number of calculated structures by all combinations_:
>
> Since the input structures are complexes, it means it leads to combine the chain A of the model 1 of the ensemble with chain B of all models?
If you are providing an ensemble of models of the complex, make sure to use only chain A for the 1st molecule and chain B for the second molecule.
And turn off the cross-dock option.
No need to multiply the number of structures - if you have 20 models in your ensemble it will generate 20 models
But if you want to refine say 10 models per input complex, then set the multiply factor to 10.