Requirements: To use AutoDock and AutoDockTools, you need to have an account in ABCC. For SGI users, please follow the instructions by skipping to the next paragraph. For PC users, who use telnet to log into their ABCC accounts, you need X-windows emulator software (ReflectionX, Exceed3D etc.) to use AutoDockTools (ADT). You can also install ADT in your PC to prepare the necessary files for AutoDock and then ftp the prepared files to your ABCC account for job submission. To visualize the autodock results, ftp back the output files from your ABCC account to your PC and use ADT to analyze the results.
The aim of this exercise is to do a mock-docking of a ligand onto a protein. For this exercise, we are going to choose an experimentally known and well-studied biotin-streptavidin complex but pretent as if the bound conformation is not known. For this exercise we will be using AutoDock to perform the docking simulation. To know more about AutoDock software, click here. At ABCC, AutoDock software is installed in SGI clusters at the location /usr/local/fbscapp/autodockroot . A local-copy of the AutoDock manual in post-script format is also kept under autodockroot (AutoDock3.0.5_UserGuide.ps).
Please log into the ncisgi server by typing the following command, ssh ncisgi and providing your username and password. To continue with this exercise, create a directory called "Docking" and change directory into the newly created Docking directory with the following command (cd Docking). I have already prepared the receptor (streptavidin) and the ligand (biotin) in PDB and mol2 (Sybyl) format respectively. The 1STP pdb entry (complex) was downloaded through Sybyl6.9 (Tripos software) and the receptor and the ligand were separated and stored in pdb and mol2 files respectively. Right-click on streptavidin and biotin (mol2 is Sybyl software format) to download the receptor and ligand structures to your hard drive.
This and the next section uses Sybyl version 6.9. To use Sybyl 6.9, type the following at the command prompt: source /usr/local/fbscapp/sybyl6.9/lib/.login
Start sybyl (sybyl version 6.9) by typing sybyl. For instructions on initilizing the software, click here. Load the protein by clicking on File >>> Read and choosing 1STPROTEIN.pdb from the file list, and click no to avoid centering the protein. Add polar hydrogens by clicking on Biopolymer >>> Add Hydrogens Click on ALL and from the Option window choose "ESSENTIAL_ONLY" to add the polar hydrogens. Block the N- and C- terminal to be charged using Biopolymer >>> Modify >> Block End and highlight ALA13 and hit OK and from the Option choose AMN to add to the N-terminal group. Again go to Biopolymer >>> Modify >> Block End and highlight VAL133 and hit OK and from the Option choose CXL to add to the C-terminal group. Now load the Kollman united charges using Biopolymer >>> Load Charges >> All >>> KOLL_UNI >>> OK . Finally save the molecule in PDB format using Prot.pdb . Please note the capitalization of the first "P" in the file Prot.pdb.
If you have already started sybyl (version 6.9), skip this paragraph. If not, type the following command source /usr/local/fbscapp/sybyl6.9/lib/.login and then type sybyl at the command prompt to start Sybyl software.
Load the ligand into Sybyl (version 6.9) using File >>> Read Biotin.mol2. Add all hydrogens using Build/Edit >>> Add >> Hydrogens . Compute partial charges using Compute >>> Charges >>> Gasteiger-Huckel To a question on "Change Formal Charges", answer no and save the molecule using File >>> Save As >> Lig.mol2 (Format:mol2)
setenv ADHOM /usr/local/fbscapp/autodockroot
setenv AUTODOCK_UTI $ADHOM/dist305/share
set path = ($path $ADHOM/dist305/bin/sgi4D.IRIX64_6.5_R10000 \
$ADHOM/dist305/share )
source /usr/local/fbscapp/adt/.adtsetup
adt
Before continuuing, if you do not go through the "Preparing the Ligand for Docking" section, please download the final ligand file by clicking here. Also make a sub-directory called "Results" for storing the results.
Ligand >>> Input Molecule >>> Read Molecule . Left click on "Files of Types" radio button and change the file format to *.mol2 . Lig.mol2 will now show up in the files list. Left-click on the Lig.mol2 Fig1 and click on Open Fig2. Now define the rigid root in the ligand molecule by left-clicking on Ligand >>> Define Rigid Root >>> Automatically . A small green sphere will be displayed showing the root of the molecule. This is usually chosen at the center-of-mass See Fig3. Then click onto the menu Ligand >>> Define Rotatable Bonds , a sub-menu will appear letting you know the number of rotatable bonds (for Biotin that would be 6) and the main window now shows the molecule rigid bonds in red and rotatable bonds in green color. Please review the default selection and modify the selection if there is any need for a change otherwise click Done . We are going to skip the Ligand >>> Aromatic Carbons menu becuase our ligand do not contain any aromatic rings. Move on to Ligand >>> Merge Non-Polar Hydrogens to merge all non-polar hydrogens onto the respective carbons. Finish up the ligand preparation by clicking onto Ligand >>> Write PDBQ... menu to create a pdbq file. Choose the filename as lig.out.pdbq See Fig4. Click here to download a copy of the lig.out.pdbq file.Before continuuing, if you did not go through "Preparing the Protein for Docking" section, please download the final receptor file by clicking here
Before this step, you can hide the ligand by going to Un/Display >>> Lines and check undisplay . Read the macromolecule by left-click on Grid >>>Macromolecule >>> Read Macromolecule Left click on "Files of Types" radio button and change the file format to *.pdb . Prot.pdb will now show up in the files list. Left-click on the Prot.pdb and click on Open This will open the Options menu. Click on "Kollman United Charges" for the charges to add. Check on yes to add solvation solvation parameters?, automatically merge non-polar Hs? and automatically merge lone pairs? . Save the result in a file called Prot.pdbqs See Fig5. Click here to download a copy of the Prot.pdbqs file. Finally, click on OK . This will save the final result in Prot.pdbqs . See Fig6. At this step you may have a zoomed-in view of the receptor, to have the full-view of the receptor, press "SHIFT-n". Set the map types by reading the formatted ligand file using the option Grid >>> Set Map Types >>> By Reading Formatted File . Left click on "Files of Types" radio button and change the file format to *.pdbq , lig.out.pdbq See Fig7. will now show up in the list of files. Left-click on "lig.out.pdbq" and hit "Open" to open the "AutoGPF Ligand" menu. Use these menus to choose the differenet atom types in your ligand and choose the polar atoms (N,O and S) to model h-bonding in docking See Fig8. Grid >>> Set Grid menu is used to set the Grid Options. The number of grids can be increased in the x,y and z directions. The spacing between the grids can also be changed but for this study we are going to keep the default value 0.375 Angstroms. Use the offset dials to move the offset box in x,y and z directions. The idea is to cover the area of interest, in this study we are interested in searching the binding area on the whole protein surface. So, we use the box to cover the whole protein. After deciding on the box size, click on File in the options menu and click on "Close Saving current" See Fig9. For Grid >>> Set Other Options use the default settings. This will make the dielectric constant distance-dependent. Use the Grid >>> Write GPF and choose a file name (Prot.gpf for example) and click on save See Fig10. Click here to download a copy of the file (Prot.gpf).
Click on Docking >>> Set Macromolecule >>> Choose Macromolecule and choose the molecule Prot . Click on Docking >>> Set Ligand Parameters >>> Choose Ligand and choose the molecule Lig_out and hit Select Ligand . This will open up AutoDPF Ligand Parameters options radio box, choose the default option for this study See Fig11. Choose Docking >>> Set Search Parameters >>> Genetic Algorithm Parameters option. If you want to try "Simulated Annealing" or "Local Search Parameters" to do your docking you can do so by clicking on respective options. Again for this study, we are going to choose the default options in the Genetic Algorithm parameters menu. Docking >>> Set Docking Run Parameters with default values See Fig12. Finally, you need to save the chosen parameters by clicking on the following option: Docking >>> Write DPF >>> GALS.dpf (GALS option is the combined Genetic Algorithm-Local Search ). Choose a file name to save the dpf parameter option, for example lig.Prot.dpf See Fig13. Click here to download a copy of the lig.Prot.dpf file.
Before you proceeed any further, make sure you have downloaded the following AutoDock input files: i) Lig.mol2, ii) Prot.pdbqs, iii) Prot.gpf and iv) lig.Prot.dpf.
PBS Pro queing system is used to submit/manage background jobs in the computational servers of the Advanced Biomedical Computing Center (ABCC). For more information on using PBS-pro in ABCC, view the manual located at http://www-fbsc.ncifcrf.gov/web_apps/pbsmanual/index.shtml . If this is the first-time you are using PBS in ABCC, type the command "prepare_for_pbs" to prepare your account for using the queing system. For more information, please read the "Before You Start section in the PBS manual. I have prepared a PBS script for this exercise, download the script from here. Open the file using the vi or any editor of your choice to see the contents of the file. Log into the ncisgi server, and submit a background job using the script by typing, qsub PBS.script at the unix command prompt. To Check the status type, qstat -an After the job is finished, go to the directory "Results" (cd Results) to see the output files. Please note that the test run was done using the default parameters of AutoDock (number of runs, energy evaluations etc.). I strongly advice you to understand each and every parameters used in AutoDock, and experiment them before you go for the final run. Also notice that even with the default values AutoDock has been fairly sucessful in locating the binding-site and the conformation of the ligand. Click here (fig.14) to see the docked (yellow) and experimental ligand (cyan) conformations for 1STP, (fig.15). You can also use the "Analyze" module of ADT to analyze the results of docking.
Got Questions or Comments, please use the contact info given belowS. Ravichandran, Ph.D. Bldg 430, Room 227 Advanced Biomedical Computing Center National Cancer Institute Frederick, MD 21702 Email: sravi@ncifcrf.gov Web: http://nciiris.ncifcrf.gov/~ravichas Voice: 301-846-1991 Fax: 301-846-5762