1. Autoligand is a script that comes with autodock tools. It has two modes: Find #n binding site within the grid region. Define shape and volume of binding site.

2. ● Step 1: Define ligand & receptor like in docking Open 4CNH_monomerFH_r eg.pdbqt Open 3U9.pdbqt as ligand

3. Step 2: Open the gridbox menu. Create Grid parameter file with 1Å spaceing. Box should be large enough to engulf the protein Step 3: Grid→Output→Save GPF… Name the new file autoligand.gpf Select: center on macromolecule Change box dimensions to 60x60x60 Change spacing to 1.000

4. Choose the.gpf you’ve created Once finished press “Launch”. Select autogrid4.exe

5. Open Compute menu Change start location to the ligand binding site Press ‘start’ to initiate Select Single fill Large sites need more points to fill

6. Ligand binds here Unexploited space

8. Select Scan Confirm to run Create 10 “fills” When script finishes load.pdb files manually. Lowest energy/vol ume 4 Unique & Novel sites

9. We’re going to search for commercially available analogs for the lead.

10. zinc15.docking.org Click here

11. Title Atom Coordina te Bond data COC1=CC=C(F)C=C1[C@@H](C)OC1=CC(=CN=C1N) C1=CN=NN1C COC1=CC=CC(C)=C1

12. Alternativel y you can draw your structure here Search for similar molecules Paste SMILES string

13. Query substances Tc=1.00 Tanimoto coefficient ZINC ID Download all results as.smi file

14. Open substances.smi with Marvin view application. Add explicit hydrogens. Clean in 3D. Save all as tripos mol2 file. Step1: Structure → Add → Explicit H atoms Step2: Structure → Clean in 3D Step 3: File ↓ Save all

16. One of many scripts written by the community. Designed to allow for virtual screening with autodock. It is an external tool and not part of autodock tools. PyRX is an alternative GUI for virtual screening, yet it isn’t free. PyRX Autodock Vina is a commandline alternative to autodock 4. It is much faster then autodock, but can also be less accurate.

17. Import substances. mol2 Proceed to receptor tab

18. Import 4CNH_monomerFH_reg.p dpqt Open options menu Keep defaults and confirm Proceed to maps tab

19. Import 4CNH.gpf Proceed to docking tab

20. Select from template

21. Import 3U9.dpf

22. Select empty directory for job files Supports threading You can select to compress the output Press ‘Generate’ to create script

23. Run this batch file

24. No simple solution in raccoon. Can load each result individually to autodock tools. Not practical for virtual screenings. Write a script that combines all the best results to a single file. Use PyRX GUI which allows for simpler visualization of screening results.

25. Autodock tools installation is straight forward. Only make sure there are no spaces or unusual characters in the path. That includes Hebrew letters! (Its better not to install it in “program files” folder) Autodock4.exe and Autogrid4.exe must be downloaded separately. Installation folder can be separate from ADT folder. ADT download: mgltools.scripps.edu/downloadsmgltools.scripps.edu/downloads Autodock download: autodock.scripps.edu/downloads/autodock- registration/autodock-4-2-download-page/ autodock.scripps.edu/downloads/autodock- registration/autodock-4-2-download-page/ Autodock vina download: vina.scripps.edu/download.htmlvina.scripps.edu/download.html

26. Download contains a raccoon.py script and raccoon.bat which runs the script. Place raccoon.py within ADT folder. raccoon.bat contains two lines: set MGLPYTHONPATH=D:\MGLTools-1.5.6 "%MGLPYTHONPATH%\python.exe“ "%MGLPYTHONPATH%\raccoon.py“ Change the path in the first line to wherever you installed ADT. In order to run RunVS.bat, you must add the directory where autodock4.exe and autogrid4.exe are located to $path environment variable, or to place these two executables inside each job directory. Raccoon download: autodock.scripps.edu/resources/raccoon autodock.scripps.edu/resources/raccoon

27. Free for academics, but login is required. Installation is entirely straight forward, haven’t encountered any issues, nor had any complaints. Marvin beans download: www.chemaxon.com/download/marvin-suite/ www.chemaxon.com/download/marvin-suite/