Example

The WnS process is shown using the example of the reference test complex of haematopoetic cell kinase (HCK, target, in green) and 1-ter-butyl-3-p-tolyl-1H-pyrazolo[3,4-D]pyrimidin-4-ylamine (PP1, ligand, in red)

Wrapper

Steps		Download files
1) Inputs Structures of the energy-minimized ligand (red) and apo target (green) molecules in Protein Databank (*.pdb) format		Input files 1qcf_ligand.pdb 1qcf_target.pdb

2) Pre-wrapper 2a) Generation of .pdbqt files with partial charges and atom types. Setting up parameter files for calculation of grid maps (.gpf) and docking (*.dpf) 2b) The docking box covers the entire target surface (red box, blind docking mode) 2c) Modification of the AD4_parameters.dat file (need to be done only once before the first wrapping cycle)		Generated files 1qcf_ligand.pdbqt 1qcf_target.pdbqt 1qcf_target.gpf 1qcf_target.dpf Modified AutoDock files AD4_parameters.dat autocomm.h

3) Wrapper cycle 1 3a) Grid maps (.map) and additional files (.glg, .fld, .xyz) are produced by Autogrid 4.2 3b) Docking log file (.dlg) is produced by AutoDock 4.2. 100 blind docking runs per cycle are performed 3c) The .dlg file is used as input by program Wrp, which first ranks and clusters the docked ligand conformations. The results are reported in a .sta file 3d) New atom types (YY, LL) of excluded atoms are assigned by wrp. The corresponding .YY.map and .LL.map files are generated before the cycle. The resulted .wrp.pdbqt file contains the docked ligand copies (cluster representatives) 3e) Available free Accessible Surface Area (ASA) is calculated by Msroll 3f) Wrapping ends if ASA ≤ 1 % or the interaction energy value of any cluster representant in the cycle is ≥ 0 kcal/mol. Otherwise, the .wrp.pdbqt file is forwarded to the next cycle 4) Wrapper cycles 2-16* The process described at Cycle 1 is repeated in consecutive cycles. Wrapping will end if a criterion of Point 3f is met. The complete Wrapping process with all files of the 16 wrapping cycles can be downloaded in a single package (O_1qcf_wrp.tgz)		Autogrid 4.2 outputs 1qcf_target.YY.map.gz 1qcf_target.LL.map.gz 1qcf_target.glg etc_grid.tgz AutoDock 4.2 output 1qcf_1.dlg Gromacs output (ver. 1.1) O_1qcf_1_ASA_free.log Msroll output (ver. 1.0) O_1qcf_1_ASA_free.log Wrp outputs O_1qcf_1.log O_1qcf_1_input_t.pdb O_1qcf_1_wrp.sta O_1qcf_1_wrp.pdbqt etc_wrp.tgz Wrapper outputs (Cycles 1-16) O_1qcf_wrp.tgz

5) Trimming After the last (16th) wrapping cycle a trimming step is involved to remove ligand copies positioned far from the target surface. The *trm.pdb file contains the target structure wrapped in a monolayer of N=143 ligand copies and can be used in Shaker.		Input files 1qcf_16_wrp.pdbqt 1qcf_ligand_template.pdb 1qcf_ligand_template.pdbqt Output file O_1qcf_16_wrp.log O_1qcf_16_wrp_trm.pdb

Shaker

Steps		Download files
6) Molecular dynamics simulation with position restraints applied on the backbone of the target protein (MD_B) The target-ligand_N (N=143) complex from the Wrapper step (O_1qcf_16_wrp_trm.pdb) is handled in a single MD_B simulation to remove a 25 % of the initial ligand copies 6a) The complex is placed in a simulation box, hydrated, and equipped with neutralizing ions 6b) Energy minimization involves steepest descend (st) and conjugate gradient (cg) runs 6c) The energy minimized complex is subjected to a 5-ns-long MD_B simulation with restraints applied on the backbone of the target protein. The resulted trajectory is stored in a portable, compressed file (.xtc) containing spatial coordinates of all atoms of all frames. The trajectories can be further processed by GROMACS tools using the corresponding binary topology (.tpr) file		Input files O_1qcf_16_wrp_trm.pdb em_st.mdp em_cg.mdp md_b.mdp Generated files posre.itp O_1qcf_16_wrp_trm_md1.tpr Output files O_1qcf_16_wrp_trm_md1.xtc

7) MD_B with simulated annealing (MD_BSA) and filtering steps 7a) Filtering 1. Migration distances and ligand-target interaction energies are calculated from the trajectories and applied for filtering out loosely bound ligand copies 7b) The remaining ligand copies bound to the target are subjected to energy-minimization, and a 20-ns-long MD_BSA (md2) 7c) Filtering 2. Migration distances and ligand-target interaction energies are calculated from the trajectories and applied for filtering out loosely bound ligand copies 7d) The remaining ligand copies bound to the target are subjected to energy-minimization, and a 20-ns-long MD_BSA (md3) 7e) Filtering 3. Migration distances and ligand-target interaction energies are calculated from the trajectories and applied for filtering out loosely bound ligand copies 7f) Clustering and ranking. As the elimination rate (ER) is ≥ 0.75, further MD_BSA & filtering steps are not necessary. Cluster representatives are collected in a final output (*_flt3_cls.pdb), and ranked by their interaction energies		Input files O_1qcf_16_wrp_trm_md1_flt1.pdb O_1qcf_16_wrp_trm_md2_flt2.pdb em_st.mdp em_cg.mdp md_bsa.mdp Generated files posre.itp O_1qcf_16_wrp_trm_md2.tpr O_1qcf_16_wrp_trm_md3.tpr Output files O_1qcf_16_wrp_trm_md2.xtc O_1qcf_16_wrp_trm_md3.xtc O_1qcf_16_wrp_trm_md3_flt3_cls.pdb

8) Refinements with flexible MD (MD_F) Target-ligand complexes are created by splitting the CC=12 cluster representatives. Each complex contains one copy of the target structure and one cluster representative. The complexes are subjected to separate 20-ns-long MD_F simulations where no restraints were applied. Example files for the first cluster are shown, and output files of all clusters are provided in a compressed library (*.tgz)		Input files O_1qcf_16_wrp_trm_md3_flt3_1.pdb em_st.mdp em_cg.mdp md_f.mdp Generated files O_1qcf_16_wrp_trm_md3_flt3_1.tpr Output files O_1qcf_16_wrp_trm_md3_flt3_1.xtc O_1qcf_16_wrp_trm_md3_flt3.tgz

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