1. Docking molecules with Vina

2. Autodock Vina

3. To study molecules they must be docked
Docked molecules bind their enzyme or receptor in a specific conformation
Docked molecules bind with high affinity
The best way to get docked conformation is to use X-ray crystallography
Alternative: use docking software like Vina to predict docked conformation

4. What is docking?
Find the conformation with which a ligand binds a receptor
This information includes full coordinates of all ligand atoms
The docked configuration is the (presumed) lowest energy binding site

5. Steps 1. get protein structure 2. create files with protein only
3. Use a known ligand (not your ligand) to define the ligand binding site
4. Generate some modified structure files
5. Use files with docking software
6. Extract predicted conformation and make new file with protein and target ligand docked

6. Make a folder
Need a place to store many files
Make c:\thesis

7. Get PDB file Go to PDB RSCB on web Search for PDB code if unknown
Once PDB code is known, download it
Use PDB/Tools, download entries and follow instructions
You must have Java enabled
Change mmCIF to PDB as type of file
Change compressed to uncompressed
Save in your thesis folder

8. PDB search

9. PDB download

10. PDB info – Protein name

11. PDB – ligand name (HET)

12. PDB coordinates

13. Start with PDB file with protein and ligand (not your ligand)
Make a copy of the PDB file
Split it into only protein and only ligand
Ligand will be used to get coordinates of binding site
Example: beta adrenergic receptor and carazolol
2rh1.pdb

14. Use wordpad text editor
Copy cau (= carazolol, ligand) from 2rh1
Paste into a file called car.pdb
REMARK carazolol
HETATM O17 CAU A O
HETATM C16 CAU A C
HETATM C18 CAU A C
HETATM N19 CAU A N
HETATM C20 CAU A C

15. PDB files have 3D structure information including XYZ coordinates of atoms
Also have atom numbers
Residue numbers
Atom types

16. Find the center of the ligand
This will be the center used when you dock your ligand to the receptor
Start with the X coordinate. Add all the X values and then divide by the number of atoms. Record for later
Repeat for Y and Z coordinates
The only purpose of having a ligand at this point is to define the ligand binding site.

17. Find coordinates z y x REMARK carazolol
HETATM O17 CAU A O
HETATM C16 CAU A C
HETATM C18 CAU A C
HETATM N19 CAU A N
HETATM C20 CAU A C

18. Carazolol – no hydrogens

19. Visualizing structures
Try rasmol or raswin
Download at google: rasmol bernstein
Several web tutorials available

20. Adding hydrogens Hydrogen is rarely found in PDB files
But all atoms are needed to dock molecules
Solution: add back missing hydrogens
We will use Babel

21. OpenBabel Download openbabel from its web site Install
Use the command prompt to run the program
Use a full ‘path’, that is a description of the program location
Add hydrogens
On my machine that is (one line):
\lm\downloads\openbabel-2.1.1\babel.exe
-ipdb car.pdb –opdb carH.pdb -h

22. Add H

23. Check that hydrogens have been added
HETATM C1 CAU C
HETATM C6 CAU C
HETATM C5 CAU C
ATOM H CAU H
ATOM H CAU H
ATOM H CAU H

24. Other ligands
Up to now we have been dealing with the ligand found in the PDB file with your receptor/protein
You may wish to try docking that ligand as a control
At some point you will want to deal with other ligands

25. Getting other ligands
To use ligands they must be transferred from the page to PDB format
Some ligands may be present in other files in the protein data bank – try searching
Ligands can be drawn, e.g. with DS Viewer Pro or other software and saved in PDB format
Most ligand files will have to have hydrogens added

26. Add hydrogen to the protein
Repeat ligand procedure, but with protein
We did car.pdb  carH.pdb
Now make copy of the protein pdb file with the ligand deleted (e.g. CAU in this example)
(you can delete WAT and other ligands too)
Now, with full path, use babel to add H

27. Add hydrogen to protein
Full path on my machine (yours will differ)
\lm\downloads\babel-2.1.1\babel.exe –ipdb bar.pdb –opdb barH.pdb -h
Result: bar.pdb  barH.pdb

28. We’re almost ready to dock
But we need two things:
Torsions
Hydrogen bonds

29. Torsions The ligand may have rotatable bonds
Benzene can not twist (0 torsions)
Hexane can twist (3 torsions, we’ll ignore rotating methyl groups)
MGLtools calculates these
Vina will twist the ligand to try to find the best fit during docking

30. H bonds We need to find H bond acceptors and donors
MGLtools also calculates these

31. MGLtools MGLtool, ADT, Autodock tools are the same
Download from MGLtools site
Install
Read tutorial
Click to open window

32. MGLtools

33. MGLtools
We will use MGLtools/Autodock tools for two things: Annotate the ligand(s) Annotate the receptor

34. Autodock / MGL tools
First we will process the ligand to add torsions and H bond information
On left side of ADT screen find ligand menu
Open ligandH.pdb (or other file)
Note: this is the ligand file you want to dock, not the X-yl structure file, unless you are docking it as a control

35. Autodock/MGLtools and ligand
Select torsion tree
Find the ligand torsion ‘root’. Detect root
Aromatic carbons: set names
Output: Save file as carH.pdbqt
It is key that you save the pdbqt file that MGLtools makes

36. ADT and protein/receptor
Find ‘grid’ menu
Macromolecule
Open receptor file with hydrogens (receptorH.pdb)
The file gets processed
Save as ligandH.pdbqt
Do not choose ‘flexible’

37. ADT receptor refinement
Now the receptor file and ligand file have been saved as .pdbqt files.
These have H bond and torsion information
Look at the ligandH.pdbqt file using wordpad or simpletext
It still looks like a .pdb file, but has extra information added

38. Now we have all of the files that we need to dock a ligand
Let’s check:
receptorH.pdbqt
ligandH.pdbqt
Binding site coordinates

39. Setting up Vina Vina needs to know what you want to do: What receptor
What ligand
Where the binding site is
Where to send out the results
This information is placed in a file called config.txt

40. Configuration file, config.txt
Config.txt gives information to Vina as it docks your ligand
The left side of each = is Vina code (don’t change)
The right side of each = is your input
You control the site of binding, the size of the site, and the receptor and ligand

41. Example Vina configuration file
out = out_carh.pdbqt receptor = 2rhe.pdbqt ligand = carh.pdbqt center_x = -28 center_y = 9 center_z = 6 size_x = 25 size_y = 25 size_z = 25 energy_range = 4

42. Download Vina Web site AutoDock Vina
Install in ‘thesis’ folder, or wherever your config file and .pdbqt files are saved.
Get the right version of Vina for your computer (e.g. PC vs. Mac)

43. Autodock Vina

44. Read manual!

45. Dock ligand Open command prompt
Make certain that command prompt is pointed to thesis directory/folder
Use the cd command with command prompt or Mac command screen

46. Finding the right directory
If necessary change directory using ‘cd’
E.g. type ‘cd \thesis’ [without the quotes] for a PC
Or type ‘cd /thesis’ [without quotes] for mac
This assumes that you put all your files in the directory ‘thesis’ on the c:drive or equivalent
Type dir/p [PC] or ls [Mac] to check that your files are present

47. Vina docking
To dock, type : vina.exe --config config.txt

48. Vina docking

49. Vina output It takes a minute or two for Vina to dock
This represents millions of docked positions being analyzed
The output file name is defined by the config file out = outfilename.txt
Each outfile has several models
We usually only care about model 1, the best

50. Output score We often want to know how tightly a ligand binds
Vina gives an estimate in the output file
Look for Vina Result: x
If x is -10 or less, binding is very tight
If x is -6 to -7 binding is just random, not tight
In between is hard to judge

51. Visualizing a docking
The easiest way is to splice the best ligand model onto the receptor with its ligand removed
Open the outfilename.txt (whatever you called it in config.txt) – the Vina output
Copy from the beginning of model 1 to model 2 [note the residue number of the ligand]
Paste into a copy of the receptor pdb file with the old ligand removed

52. Seeing is believing
Now you can open your new file with receptor and docked ligand using RasWin or another visualization program
Use the residue number of the ligand to select it (e.g. select 480)
Convert to spacefill (e.g. spacefill)
How does it look? Is it in a binding pocked?

53. Next steps Having a ligand binding model is valuable
Specific contacts can be analyzed
By comparing two or more models and the contacts that they make, patterns of receptor change can be determined
E.g differences between active and inactive receptors or receptor subtypes can be understood