The scripts and input files that accompany this demo can be found in the demos/public directory of the Rosetta weekly releases.

KEYWORDS: DESIGN METALS INTERFACES ENZYMES

This is a demo for the mononuclear zinc metalloenzyme redesign procedure described in Khare, Kipnis, Greisen et al. Nature Chemical Biology (2012).

Authors: Sagar Khare (khares@uw.edu), Per Jr Greisen (pgreisen@gmail.com) Edited by Vikram K. Mulligan (vmullig@uw.edu) during the 2016 Documentation XRW.

## Requirements

The scripts in this demo require that SciPy is installed on the user's system.

## Summary

Starting from a TS ensemble model and a set of zinc-containing PDBs (only one - PDBid 1A4L - is included here, list of other PDB codes used in the paper is in list_of_input_pdbs file) as inputs, we generate a design model and evaluate it.

## Input

Requires an input TS model including the metal, and a (set of) PDB file(s). The TS ensemble is generated using a starting molfile, and converted to Rosetta parameters using the script ~/Rosetta/main/source/src/python/apps/public/molfile_to_params.py.

## Overview of the Procedure

Each Step is included as a subdirectory with a separate README. In each case, the relevant command is intended to be run from the relevant subdirectory.

1. Analysis of ZincSite
2. CleanPDB after running the zinc analysis.
3. Align Transition State model(s) to the Zinc
4. Minimization in polyAla pocket (optional)
5. Matching to introduce additional catalytic residues
6. Design to maximize TS affinity
7. Revert to Native - Semi-automated refinement
8. Dock (validation)

## Details

For most Python scripts, running with a -h option will give a list of available options. To run the commands shown below, you will either need to set your ROSETTA_TOOLS environment variable to point to the Rosetta/tools directory, or you will need to replace $ROSETTA_TOOLS with the path to your Rosetta/tools directory manually. (To set the environment variable temporarily, for the current session, use ROSETTA_TOOLS=<path_to_Rosetta_tools_directory>. For example, if your Rosetta tools directory were /programs/Rosetta/tools/, use ROSETTA_TOOLS=/programs/Rosetta/tools. Each of the following commands is intended to be run from the relevant subdirectory for the step. For example, before running the first command, navigate to the 1_Analyze_ZincSite subdirectory. 1. Given a PDB file, obtain the co-ordination sphere details of metal (how many protein-metal, and HETATM-metal interactions, in what geometry in each chain). python$ROSETTA_TOOLS/zinc_site_redesign/analyze_zinc_site.py -f 1A4L.pdb
2. Given an input PDB, "clean" it i.e. keep one chain, change MSE->MET, KCX->LYS etc. Keep HETATMS.

python ROSETTA_TOOLS/zinc_site_redesign/cleanPDBfile.py -f 1A4L.pdb 3. Align transition state model(s) onto the zinc site according to co-ordination sphere details: 1. Given TS model (LG_0001.pdb) and "clean" PDBfile (1A4L_clean.pdb) align them. pythonROSETTA_TOOLS/zinc_site_redesign/align.py -f 1A4L_clean_A.pdb -l LG_0001.pdb
2. Generate Rosetta Inputs given the aligned ligand.

python ROSETTA_TOOLS/zinc_site_redesign/generate_metal_cstfile.py -f 1A4L_clean_A.pdb -m ZN -a aligned_ligand.pdb 4. Minimize the constraints energy for the superimposed ligand using Rosetta. Except the metal-chelating protein residues, every other ligand-proximal protein residue is temporarily converted to Ala. <path_to_Rosetta_directory>/main/source/bin/enzyme_design.default.linuxgccrelease @optcst.flags -linmem_ig 10 -in:file::s rosetta_cst.pdb Note that, in the above, you may need to change "default.linuxgccrelease" to match your build, operating system, and compiler (e.g. static.macosclangrelease). 5. To introduce additional catalytic residues, run a round of RosettaMatch. <path_to_Rosetta_directory>/main/source/bin/match.default.linuxgccrelease @general_matching.flags @scaf.flags @subs.flags -linmem_ig 10 -in:file::s 1A4L_clean_A_r.pdb 6. Design the rest of the pocket for maximizing TS affinity. <path_to_Rosetta_directory>/main/source/bin/enzyme_design.default.linuxgccrelease @enzdes.flags -correct -linmem_ig 10 -in:file::s <file_from_matching.pdb> > design.log & 7. For a semi-automated refinement step, generate a so-called resfile, so that the wildtype and any other user-specified residue can be introduced at any given position. Use this resfile with similar commandline as Step 6. pythonROSETTA_TOOLS/zinc_site_redesign/generate_residuefile.py UM_1_H15H17H214D295Q58_1A4L_clean_A_r_1A4L_clean_A_1__DE_1.pdb
1. To validate the design, perform docking of the ligand in the designed pocket to check if there are any alternative binding modes with similar energies.
<path_to_Rosetta_directory>/main/source/bin/ligand_dock.default.linuxgccrelease @flags`