Problem with protein and ligand preparation before ligand docking.

So the issue you’re having with missing residues/gaps is that there are certain residues in your input PDB which have backbone atoms with zero occupancy. This is likely because the electron density in those regions is too weak to see, but the crystallographer could be pretty certain of the coordinates based on the surrounding atoms. The issue here is that, by default, Rosetta will ignore residues with missing & zero occupancy backbone atoms. As such, the clean_pdb script will throw those atoms out. (That’s actually one of the things it’s explicitly “cleaning”.)

One fix might be to open the input file in a text editor and manually change the “0.00” in the occupancy columns of ASP 31 and GLY 34 to be “1.00”. Another option may be to provide the `–keepzeroocc` flag to the clean_pdb.py script, and then pass `-ignore_zero_occupancy false` to the Rosetta application command line.

A third option is to skip the clean_pdb step altogether. Rosetta is much more robust to reading in PDBs than it was in the past, so cleaning is often not necessary. You may want to edit the PDB to remove extra chains and/or some of the crystallization buffers (e.g. the phosphates), but Rosetta should be able to read in the PDB directly without cleaning. (Though you will probably still want to pass `-ignore_zero_occupancy false` in your case.)

Regarding prep, doing a pre-relax probably won’t hurt. But I’d recommend that you add the flags `-constrain_relax_to_start_coords` and `-relax:ramp_constraints false` to your relax command line to keep the backbone from moving too much. (You could also constrain the sidechains, but as ligand docking allows the sidechains to move, it probably isn’t necessary.)

Regarding the double bonds, the PDB format strictly speaking doesn’t actually have any bond order information. There’s some conventions which can be used to represent it, but that depends on the program reading it to recognize those non-standard addtions. Rosetta does not. Instead, the presence/abscense of double bonds is driven entirely by the designation in the params files. If you have the proper single/double annotations in the Mol file you pass to molefile_to_params.py, then the params file will have the correct bonding, and Rosetta will properly recognize the bonds — even if there’s no such annotation in the PDB file.