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|DI-UBIQUITIN TUTORIAL FOR THE HADDOCK WEBSERVER|
|USING RDC AND CSP DATA                         |
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This tutorial file contains a description of the di-ubiquitin docking 
case as was approximately performed in (1). It uses the GURU interface
of the HADDOCK webserver (haddock.science.uu.nl/Haddock). 

     */Note/* The data used for the docking comes from CSP and RDC NMR
              experiments (2).
              A paper describing the tutorial and docking case can be 
              found in (3). 

This file contains the following sections:
    1. Contents
    2. Setting up the HADDOCK webserver parameters
     * References

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*1.* *_Contents_*

The tutorial directory contains three folders:

    pdbs/       : It contains the structures used for docking. 
                  For the webserver only the 1AAR_1D3Z_ensemble.pdb 
                  is of importance. 

                  Files: 1AAR_[1-2].pdb
                         1D3Z_0[01-10].pdb
                         1AAR_1D3Z_ensemble.pdb

    data/       : Data files used to create the HADDOCK/CNS restraints.
                  
                  Files: active-passive_residues.data
                             a list of the active and passive residues
                             for each chain. 

                         alignment_tensor_pc.data
                             the principal components of the alignment
                             tensor used to create the VEAN restraints.
                             also holds the R and D values used during
                             docking.

                         ubiDP_rdc_segid.data
                             the RDC value for each residue on each
                             chain. 

    restraints/ : The HADDOCK/CNS restraint files.   
                  
                  Files: ubiD-ubiP_airs.tbl    
                             defines the AIR restraints, based on the 
                             active and passive residues.

                         ubiD-ubiP_pepbond.tbl 
                             unambiguous interaction restraints, which
                             represent the isopeptide bond.

                         ubiDP_vean_inter.tbl  
                             intermolecular VEAN restraints.

                         ubiDP_vean_intra.tbl  
                             intramolecular VEAN restraints.

                         UbiDP_sani.tbl        
                             direct SANI restraints.

    haddockparam-di-ubiquitin.web: this is an example of a paramter file that can be downloaded 
                                   from the web server after a successful submission. This file can be
                                   uploaded in the file upload interface of the web server.

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*2.* *_Setting up the HADDOCK webserver parameters_*

1. Go to the HADDOCK webserver located at haddock.science.uu.nl/Haddock
and open the GURU interface. 

2. Give the docking run a name (di-ubiquitin_CSP_RDC).

3. Expand the **First molecule** section. Set _Where is the structure 
provided?_ to _I am submitting it_. Set _Which chain of the structure
must be used?_ to _A_. Click on the _Browse..._ button next to _PDB 
structure to submit_ and select the _1AAR_1D3Z_ensemble.pdb_ file.

4. Next to _Active residues_ fill in the list of residue numbers as given
in /*active-passive_residues.data*/ file for the first molecule. Expand
the *Fully flexible segments* section. Set _First number_ of Segment 1
to 72 and _Last number_ to 76. Uncheck the box _The C-terminus of your
protein is negatively charged_. 

5. Expand the **Second molecule** section. Set the _Structure definition_
parameters indentical as in the **First molecule** section. Fill in the
_Active residues_ and _Passive residues_ as given in 
/*active-passive_residues.data*/. Expand the *Fully flexible segments* 
subsection. Set for Segment 1 _First number_ and _Last number_ 
to 48 and for Segment 2 to 72 and 76 respectively. 

6. Expand the **Distance restraints** section. Click on the _Browse..._ 
button next to _You can supply a HADDOCK... (unambiguous restraints)_ 
and select in the restraints folder /*ubiD-ubiP_pepbond.tbl*/. Uncheck
the _Randomly exclude a fraction of the ambiguous restrants (AIRs)_ 
box. 

7. Expand the **Sampling parameters** section and set _Number of 
structures for rigid body docking to 1440. 

8. Expand the **Restraints energy constants** section. Expand the *Energy
constants for unabmiguous restraints* subsection. Set _hot_, _cool1_, 
_cool2_ and _cool3_ to 0.1, 1, 5 and 5 respectively.

9. Expand the **Residual dipolar couplings** section. Set _RDC type_ to 
VEAN in the *Residual dipolar couplings 1* subsection. Expand the 
-SANI energy constants- subsubsection and set _Last iteration_ to 1. 
Click on _Browse..._ in the -VEAN energy constants- subsubsection and 
select in the restraints/ folder /*ubiDP_vean_inter.tbl*/. Expand 
*Residual dipolor couplings 2*. Set _RDC_type_ to VEAN. In the -SANI 
energy constants- subsubsection set _First iteration_ and _Last 
iteration_ to 1. Set the _RDC file_ to /*ubiDP_vean_intra.tbl*/. Expand 
the *Residual dipolar couplings 3* subsection. Set _RDC type_ to SANI 
and _R_ and _D_ to 0.057 and -11.49 respectively. In the -SANI energy 
constants- subsubsection set _First iteration_ and _Last iteration_ to
2. Upload as _RDC file_ /*ubiDP_sani.tbl*/

10. Go to the bottom of the screen and enter _Username_ and _Password__. 
Submit the docking run by pressing _Submit Query_. 

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*_References_*

1. van Dijk A.D.J. /et al./ (2005) /Proteins/ *60*, 367--381
2. Varadan R. /et al./ (2002) /J.Mol.Biol./ *324*, 637--647
3. van Zundert G.C.P. /Bonvin A.M.J.J. / (2014) /Methods Mol. Biol./ **, 163--179
