Setting up the INPHARMA run

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The next step is to configure your INPHARMA run. To avoid a mess of files we will generate a designated directory for this particular pair of ligands:

cd 07_inpharma

mkdir 01_LL1-M77
cd 01_LL1-M77

The following configuration file is all you need to set up your INPHARMA run:

Configuration file

{
  "Assignment": {
    "Ligand1": "../../04_asgn/LL1_M77.asgn", 
    "Ligand2": "../../04_asgn/M77_LL1.asgn"
  }, 
  "HList": {
    "Ligand1": "../../03_hlist/LL1.hlist", 
    "Ligand2": "../../03_hlist/M77.hlist", 
    "Receptor": "../../03_hlist/receptor.hlist"
  }, 
  "InOut": {
    "Corr_Output": "corr.out.dat", 
    "Corr_Output_Mode": "all", 
    "Experimental_Peaklist": "../../16_vols/LL1-M77.vols", 
    "Fit_Quality_Output": "qual.dat", 
    "Input_Directory": ".", 
    "Output_Directory": "out", 
    "Verbose": "off", 
    "Vol_Output": "vol.dat", 
    "Vol_Output_Mode": "norm"
  }, 
  "Kinetic": {
    "Model": 2, 
    "kL1": 300000.0, 
    "kL2": 100000.0
  }, 
  "Relaxation": {
    "Correlation_Time_Complexes": 15.0, 
    "Correlation_Time_Ligand1": 0.1, 
    "Correlation_Time_Ligand2": 0.1, 
    "Cutoff": 8.0
  }, 
  "Structures": {
    "Directory_Complex1": ".", 
    "Directory_Complex2": ".",
    "Fileformat": "pdb", 
    "List_Complex1": "modes_LL1.nam.all",
    "List_Complex2": "modes_M77.nam.all",
    "Topology_Complex1": "../../02_structures/3DNE_LL1.pdb", 
    "Topology_Complex2": "../../02_structures/3DNE_M77.pdb"
  }
}

The first part of the configuration file links to the appropriate INPHARMA files we have generated in earlier steps. This should be relatively straightforward if you have built your filesystem as suggested in this tutorial.

The arguments Struct_List_Complex1 and Struct_List_Complex2 should point to a file containing file names of docking modes. These files can be generated by:

ls ../../01_prepare_structures/01_3DNE_LL1/02_docking/pdbs_min/*.pdb > modes_ligA.nam
ls ../../01_prepare_structures/02_3DNE_M77/02_docking/pdbs_min/*.pdb > modes_ligB.nam

Note: Remember that each protein-ligand complex must have atom numbering in agreement with Complex1_Pdb and Complex2_Pdb, and associated hlist and asgn files.

How can I get those parameters

  • Spectrometer_Frequency: Larmor Frequency of the spectrometer you recorded your INPHARMA spectra.
  • Correlation_Time_of_LXFree: correlation time of your ligands this value can be obtained experimentally
    • using T1-Inversion recovery experiments
    • T2-CPMG experiments
  • Correlation_Time_of_Complexes: correlation time of your complex, it can be obtained
    • theoretical estimate (if you have a structure of the complex) can be computed with HydroNMR [1]
    • experimentally carefully recording T2 jump-return experiments
  • Kinetic_Model: your kinetic model assumption according to Orts et al. Angewandte 2009 [2]
    • 2 means you assume the ligands are in molar excess and your target always bound to the ligands. In this case kL1 and kL2 are the "swapping" constants of L1 and L2. For the fast exchange assumption to be valid values large than ~10000 are needed for these parameters. Thus, these should be chosen proportional to the ratio of the ligands' kd values multiples by a scaling factor (e.g. 1000). The exact scaling factor is not important as long as the values are in the valid range.
      • kL1 300000.0
      • kL2 100000.0
    • 1 means you assume that the target can be also free (see also the SpINPHARMA section)
  • Concentrations
    • cL1_tot: total concentration of ligand 1 in micromolar scale
    • cL2_tot: total concentration of ligand 2 in micromolar scale
    • cT_tot: total concentration of the target in your experimental setup
    • you can estimate this by UV spectroscopy [3] or Bradford assay [4]