I am trying to complete a science fair project. I want to use FoldX and Boltz2 to find a stable mutation of PETase with cold affinity. I think I can do this by running FoldX to find stability (DDG) and Boltz2 for cold affinity on relevant mutations suggested by ChimeraX.
Specifically, by giving FoldX the 6eqe.pdb for PETase and running the RepairPDB and RunModel commands on all the different mutations of interest, I should get all their DDGs so that I can use the most stable ones for further study of cold affinities in Boltz2.
However, I am unable to get FoldX working properly. This is embarrassing because it's nearly the first step in the flow, and there are several papers written with Fold-X, so I know it must work. But, neither I, my mentor, nor my engineering father can figure it out. Any help, guidance, or mentorship would be much appreciated.
What doesn't work
I have 4 problems:
Version doesn't work. I can't seem to get the basic installation correct if the version doesn't even work, right?
"Repaired" PDB is missing TER/END statements.
BuildModel:
_FOLDXDEFINED
error and wrong/no mutation implemented.
Output message:
Sorry yo specified an option_FOLDXDEFINED for the order of mutations that does not exist, should be _USERDEFINED or _FOLDXDEFINED
No mutation applied (6EQE) or the wrong mutation is made (1UBQ: LEU at pos 8 mutated to ALA instead of VAL).
- PositionScan: terminated:
Specified residue not found
for A8, empty PS_*.fxout.
What I did
- Install FoldX 5.1
Download from the link sent after registration as a high school student - https://foldxsuite.crg.eu/system/files/foldx5_1Linux64.zip
Install it:
cd ~/Downloads
unzip foldx_1Linux64_0.zip -d /home/isef/proj/isef/tools/foldx/
cd /home/isef/proj/isef/tools/foldx/
chmod +x foldx_20251231
ls -l
total 199448
-rwxrwxr-x 1 isef isef 86720392 Apr 17 04:11 foldx_20251231
-rwxrwxr-x 1 isef isef 114894632 Apr 17 03:59 foldx4_20251231
drwxr-xr-x 2 isef isef 4096 Feb 23 2024 molecules
-rw-r--r-- 1 isef isef 2573870 Nov 15 2021 rotabase.txt
-rw-rw-r-- 1 isef isef 36791 Apr 17 03:59 yasaraPlugin.zip
Verify the binary ``` ./foldx_20251231 -v 2>&1 | tee -a /tmp/foldx_version_log
FoldX 5.1 (c)
code by the FoldX Consortium
Jesper Borg, Frederic Rousseau Joost Schymkowitz, Luis Serrano Peter Vanhee, Erik Verschueren Lies Baeten, Javier Delgado and Francois Stricher and any other of the 9! permutations based on an original concept by Raphael Guerois and Luis Serrano
*** THIS IS AN IMPORTANT MESSAGE:
External parametrized molecules were detected... Final energetic values will depend of the correct parametrization of this molecules.
the required argument for option '--version' is missing
3. **Run RepairPDB and check repaired file**. I started with 6eqe.pdb, my target enzyme, but that didn't work at all. By didn't "work at all", I mean that in addition to the 1UBQ errors to follow, it didn't perform any mutations at the desired position and reported 0s for total energy for all but the first run when given numberOfRuns > 1.
So, I asked AI for the simplest possible mutation that FoldX can reliably do. It suggested mutating LEU at position 8 to VAL in 1ubq.pdb, so that's where I am focusing my effort. I just want to get 1 mutation to work correctly and then I can expand to my needs in 6eqe independently.
cd /tmp/foldx_temp wget https://files.rcsb.org/download/1UBQ.pdb -O 1ubq.pdb /home/isef/proj/isef/tools/foldx/foldx_20251231 --command=RepairPDB --pdb=1ubq.pdb --out-pdb=true 2>&1 | tee repair.log grep -E "TER|END" 1ubq_Repair.pdb
The end of the repair says
End of Repair Output PDB ./1ubq_Repair.pdb Your file run OK End time of FoldX: Mon Oct 6 19:32:47 2025 Total time spend: 35.80 seconds. Cleaning RepairPDB...DONE
so I get my hopes up that it worked. Unfortunately, there are no TER or END in the repaired file.
1. **Confirm LEU is at position 8 in the Amino Acid**
$ awk '$6==8 {print}' 1ubq_Repair.pdb | head ATOM 59 N LEU A 8 30.132 40.069 18.642 1.00 9.84 ATOM 60 CA LEU A 8 29.607 41.180 19.467 1.00 14.15 ATOM 61 C LEU A 8 30.075 42.538 18.984 1.00 17.37 ATOM 62 O LEU A 8 29.586 43.570 19.483 1.00 17.01 ATOM 63 CB LEU A 8 29.919 40.890 20.938 1.00 16.63 ATOM 64 CG LEU A 8 28.876 40.104 21.721 1.00 18.88 ATOM 65 CD1 LEU A 8 28.679 38.712 21.146 1.00 19.31 ATOM 66 CD2 LEU A 8 29.258 39.960 23.190 1.00 18.59
So, the 1ubq_Repair.pdb looks like it should at position 8.
5. **Run BuildModel**
5a. **Create the individual_list.txt and BuildModel.cfg**
cd /tmp/foldx_temp echo "LA8V;" > individual_list.txt cat > BuildModel.cfg << EOF command=BuildModel pdb=1ubq_Repair.pdb mutant-file=individual_list.txt numberOfRuns=1 out-pdb=true EOF
5b. **Run the BuildModel and save the log**
/home/isef/proj/isef/tools/foldx/foldx -f BuildModel.cfg 2>&1 | tee /tmp/foldx_log
Produced the following tail of output ...
Starting BuildModel Reading LA8V; Residue to Mutate LEUA8 has residue index 7 Sorry yo specified an option_FOLDXDEFINED for the order of mutations that does not exist, should be _USERDEFINED or _FOLDXDEFINED Sorry yo specified an option_FOLDXDEFINED for the order of mutations that does not exist, should be _USERDEFINED or _FOLDXDEFINED Your file run OK End time of FoldX: Mon Oct 6 20:02:17 2025 Total time spend: 0.40 seconds. validated file "1ubq_Repair_1.pdb" => successfully finished Cleaning BuildModel...DONE
5c. **Check to see if the mutation was done**
awk '$6 == 8 {print $0}' /tmp/foldx_temp/1ubq_Repair_1.pdb ATOM 59 N ALA A 8 30.132 40.069 18.642 1.00 9.84 ATOM 60 CA ALA A 8 29.607 41.180 19.467 1.00 14.15 ATOM 61 C ALA A 8 30.075 42.538 18.984 1.00 17.37 ATOM 62 O ALA A 8 29.586 43.570 19.483 1.00 17.01 ATOM 63 CB ALA A 8 29.919 40.890 20.938 1.00 16.47
It's not LEU anymore, so that's good; however, it's ALA instead of VAL, which is not good.
5d. **Check the output**
$ cat Dif_1ubq_Repair.fxout FoldX 5.1 (2011) by the FoldX Consortium Jesper Borg, Frederic Rousseau, Joost Schymkowitz,
Luis Serrano and Francois Stricher
PDB file analysed: batch Output type: BuildModel Pdb total energy Backbone Hbond Sidechain Hbond Van der Waals Electrostatics Solvation Polar Solvation Hydrophobic Van der Waals clashes entropy sidechain entropy mainchain sloop_entropy mloop_entropy cis_bond torsional clash backbone clash helix dipole water bridge disulfide electrostatic kon partial covalent bonds energy Ionisation Entropy Complex 1ubq_Repair_1.pdb 0.998679 0.00173548 0 0.867633 0 -0.917758 1.44122 -4.44089e-16 -0.361689 0.00608543 0 0 0 -0.0385523 -1.20861e-06 0 000 0 0 0
So, it seems to have produced total energy, but is this for that VAL mutation or the ALA mutation?
6. **Run PositionScan**. Maybe we can figure get it with PositionScan by getting all the mutations at position 8 since specifying the mutation didn't work.
6a. **Run it**
/home/isef/proj/isef/tools/foldx/foldx --command=PositionScan --pdb=1ubq_Repair.pdb --positions=A8 --output-file=pos_scan 2>&1 | tee posscan.log
Relevant tail of the output:
Specified residue not found. no mutations performed. Your file run OK End time of FoldX: Mon Oct 6 20:13:45 2025 Total time spend: 0.60 seconds. Cleaning PositionScan...DONE
6b. **Check the output**.
$ cat pos_scan terminated
6c. **Are there any more files of note?** At the bottom of an `ls -ltr` there were only 3 files:
-rw-rw-r-- 1 isef isef 0 Oct 6 20:13 PS_pos_scan_scanning_output.txt -rw-rw-r-- 1 isef isef 11 Oct 6 20:13 pos_scan -rw-rw-r-- 1 isef isef 1804 Oct 6 20:13 posscan.log ```
- Make logs available
- repair.log for 1ubq
- 1ubq_Repair.pdb
- foldx_log for 1ubq_Repair.pdb
The program is definitely working. It doesn't matter that it didn't produce
TER
andEND
lines at the end. Those are optional in PDB files.For the rest, it is almost impossible to read this as the whole thing is improperly formatted. If you put three backtick signs (like this `, on the same key in the upper left corner as ~ or tilde) as a single line at the start of the output you want to format, and the same three backtick signs as a single line at the end, it will be formatted like this:
Someone might be able to help from a properly formatted post.
By the way, a simple Google search will give you a clear clue how to fix this:
https://www.google.com/search?q=Sorry+you+specified+an+option_FOLDXDEFINED+for+the+order+of+mutations+that+does+not+exist%2C+should+be+_USERDEFINED+or+_FOLDXDEFINED&sca_esv=1e4238bc8dc008dd&rlz=1C1UEAD_enUS1016US1016&ei=Fv7laPewF6WQm9cPtaOg6QE&ved=0ahUKEwj3k8TC9pOQAxUlyOYEHbURKB0Q4dUDCBA&uact=5&oq=Sorry+you+specified+an+option_FOLDXDEFINED+for+the+order+of+mutations+that+does+not+exist%2C+should+be+_USERDEFINED+or+_FOLDXDEFINED