Reply To: I’m a total beginner to Rosetta 3.2 and there are no tutorials for me to follow. Can someone help.? =)

[Anonymous]

Overview and checklist
Here is a “checklist” for setting up a ligand docking experiment. Details for all the steps are given below.
A. Reformat starting structures
1. Protein receptor in PDB format with no waters or cofactors; each metal ion should have a unique chain ID.
a. Does this mean that from the PDB file downloaded, we just need to delete the waters and metal ions, and just keep the protein residues instead?

Delete everything that’s not a protein atom. Rosetta ignores all the stuff at the beginning, may as well delete it too…

2. Cofactors, if any, should be in .sdf or .mol2 format, with coordinates taken from the original PDB file.
a. Does this mean that I should make a copy of the original PDB file, then delete all the lines just leaving the cofactor (metal ion), and then convert to sdf or mol2 using Babel?

I guess so. PyMOL can do it too.

3. Ligands to be docked should be in .sdf or .mol2 format, one ligand per file. If you’re trying to reproduce a known binding mode, those ligand coordinates should be used.
a. Does this mean that I should make a copy of the original PDB file, then delete all the lines just leaving the ligand, and then convert to sdf or mol2 using Babel?
B. Prepare ligands and cofactors
1. Generate conformers if desired. All conformers should be in the same .sdf / .mol2; one file per compound. Tautomers / protomers count as different compounds.
2. Assign partial charges if desired. Only charges from the first entry in the file are used. Charges can only be read from .mol2 files.
a. If I do not have Openeye, does that mean I –skip-omega and –skip-charges?

I have no idea what you’re asking, sorry…

3. Convert the .sdf / .mol2 into a .params file and one or more .pdb files. The .params describes atom types, charges, and rotatable bonds; the .pdb files contain ligand coordinates.
4. Make a ligand conformer library if desired. Concatenate the desired coordinates in PDB format and add a “PDB_ROTAMERS …” line to the .params file.
C. Prepare the receptors
1. Repack the entire receptor in the presence of cofactors but the absence of ligands.
a. Here, when we use ligand_rpkmin.linuxgccrelease, do we use it on the ligand PDB or the protein PDB?

protein

2. Choose the lowest-energy result to use as the repacked structure.
3. I repack by running “relax” with the sequence option.
D. Generate input files for Rosetta
1. Ligand .params files
2. Ligand conformer libraries (typically *_confs.pdb)
3. Input PDB: repacked protein + generated ligand conformation
4. Native PDB: experimental protein and ligand coordinates (optional, for RMSD calculations)
5. Unbound PDB: experimental protein without ligand (optional)
6. Rosetta flags file (typically FLAGS.txt)
E. Run docking trajectories
F. Analyze results
1. Concatenate silent.out files to get one per ligand/receptor pairing.
2. Extract score and RMSD information; make scatterplots.
3. Cluster ligand conformations.
4. Estimate docking confidence.