A precompiled executable can be found in the bin folder. If you wish to recompile the code, e.g. after making some modifications, you can recompile it using the GNU fortran compiler thus:
gfortran src/analyze_alphafold2_metal_clusters_for_release.f -o analyze_alphafold2_metal_clusters_for_release.exe
The code requires that a total of nine(!) command line arguments be provided by the user. They are described below in the order that they are required; the values used in our paper are provided in parentheses. All distances listed are in Angstroms. A variety of information is written to the screen (and can be piped to an output file if desired).
analyze_alphafold2_metal_clusters_for_release.exe input.pdb ligand_list_file regionDistanceMinimum regionDistanceMaximum stericClashCutoff disulfideCutoff addBbnAtomCutoff ligandLigandClashCutoff jobID
In order, the arguments are:
input.pdb = A file containing the protein of interest in PDB format
ligand_list_file = A file listing all of the ligand types to search for, together with their RMSD cutoffs
regionDistanceMinimum = (0.0) "regions" are merged if any pair of atoms are further than this distance and...
regionDistanceMaximum = (8.0) ...no greater than this distance...
stericClashCutoff = (2.0) reject ligand placements if any ligand atom is within this distance of a protein atom
disulfideCutoff = (2.5) SG atoms in cysteines are considered disulfides if within this distance
addBbnAtomCutoff = (0.0) backbone atoms, e.g. carboxyl oxygens, are added to a "region" if within this distance
ligandLigandClashCutoff = (2.5) reject ligand placements if any ligand atom is within this distance of a previously placed ligand atom
jobID = (999) an integer to identify this job if multiple jobs are run in the same folder simultaneously
The format of the ligand_list_file should be as follows
PDB-FILE-NAME NUMBER-OF-ATOMS-FOR-SUPERPOSITION RMSD-CUTOFF
For example:
4Fe4S_4CYS.pdb 4 0.5
An example run, including making a temporary directory, downloading an AlphaFold2 PDB file, and copying the contents of the TEMPLATES folder, would be performed as follows:
mkdir TMP
cd TMP
wget https://alphafold.ebi.ac.uk/files/AF-P08201-F1-model_v1.pdb
cp ../TEMPLATES/* .
analyze_alphafold2_metal_clusters_for_release.exe AF-P08201-F1-model_v1.pdb ligand_list_FES_ZINC_RMSD_0.5_12_LIGANDS 0.0 8.0 2.0 2.5 0.0 2.5 999
The code generates several output files, most of which were used in debugging the code. The most critical files are as follows:
ligand_summary_info_XXXXXX.txt = Contains information on each ligand type placed, including the coordinating residues, pLDDT scores, RMSDs, etc
ligand_sites_XXXXXX.pdb = A PDB file containing the superimposed coordinates of all placed ligands - this can be combined with the ATOM entries from the original AlphaFold2 pdb file
The XXXXXX in the files above corresponds to the jobID input argument.
The code was run on the 21 proteomes that were predicted with AlphaFold2. The results of this effort can be found in the DATA folder, which contains both a single excel file (LIGAND_BINDING_SITES_SUMMARY/ligand_binding_sites_summary.xlsx) with summary information about each ligand binding site that we identified, and separate folders for each organism containing a .tar file combining all of the ligand_sites_XXXXXX.pdb files.
Please cite the following work if you use our code or our data:
Wehrspan ZJ, McDonnell RT, Elcock AH. Identification of Iron-Sulfur (Fe-S) Cluster and Zinc (Zn) Binding Sites Within Proteomes Predicted by DeepMind’s AlphaFold2 Program Dramatically Expands the Metalloproteome. Journal of Molecular Biology. 2022;434(2):167377. https://doi.org/10.1016/j.jmb.2021.167377 pmid:34838520