Tutorial

DockoMatic: Automated Tool for Homology Modeling and Docking Studies

DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Learning Objectives

I. Create & validate a protein homology model

II. Use molecular docking to calculate the binding energy for a drug to the protein
III. Assessment of docking results IV. Visualize the protein-drug complex

DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Systems of Interest
 Protein:
 223

Collagen α1 (XI) NPP domain

amino acid protein  2 disulfide bonds  A minor fibrillar collagen involved in thrombosis
 Drug:
A

Arixtra (a.k.a. Fondaparinux)

heparin-based drug  An anticoagulant used to treat blood clots  Binds Col α1 (XI) NPP to prevent blood -O SO 3 coagulation
Arixtra structure
-O3SO O
OH OH OH

-O3SO O O O HN OH O
OSO3-

O OH O O
COOOH

O

O CH3 HN SO3-

O OSO3OSO3-

O HN

SO3-

DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

DockoMatic 2.0

User friendly graphical user interface (GUI) for automation

Homology model creation using the Timely Integrated Modeller (TIM) interface Create, submit, manage AutoDock1 molecular docking calculations Track jobs & analyze results using PyMol2

1. Morris, G. M. et al. J. Computational Chemistry 2009, 16: 2785-91. 2. The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC.

DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking . 112. Biol. 535. (1977) J.How many structures solved? The Protein Data Bank* Dposition Year 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 *Bernstein. Mol. X-Ray Structures 971 1148 1503 1793 2149 2457 2657 2782 3917 4454 5131 5664 6695 6115 7357 7790 8308 3953 Total X-Ray Structures 971 2119 3622 5415 7564 10021 12678 15460 19377 23831 28962 34626 41321 47436 54793 62583 70891 74844 et al.

Part I: Homology Modeling DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

ligand binding paradigms in drug design.Homology Modeling: Overview  Prediction of 3D-structure of a given protein sequence (target) based on alignment to known protein structures (templates)  A useful model requires 30% sequence identity between target and template Models used to understand function.  DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking . etc. activity.

W5-9.Homology Model: Template Selection 1. Begin with the target amino acid sequence Use the Basic Local Alignment Search Tool* (BLAST) to search for amino acid sequences of template proteins where the 3D structure is available The principle repository for 3D protein structures is the National Center for Biotechnology Information+ (NCBI) Only amino acid sequences with greater than 30% identity between target and template should be considered. 3. *Johnson et al. (2002) Nucleic Acids Res. +Edgar. 2. et al. 36. 4. 30. look to protein templates with similar functionality as the target. (2008) Nucleic Acids Res. 5. 207-210 DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking . If functionality is known for a class or family of proteins.

Homology Modeling: Flow Chart Known (Template) Structures Target Sequence Template Selection NO Evaluate the Model YES Homology Model Alignment (TargetTemplate) Model Building for the Target DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

Homology Model Creation: DockoMatic Procedure DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

• Open Terminal Window DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

• Type: ssh –X username@host DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

• Enter password DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

• This is the HOME directory DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

• Type: cd $SCRATCH DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

• • • • Type: mkdir DOCKOMATIC-WORKSHOP Type: cd DOCKOMATIC-WORKSHOP Type: dockomatic DockoMatic will now open in a new window DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

DockoMatic 2.0 GUI DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

DockoMatic Select: Use Modeller  DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

DockoMatic: Accessing the TIM Wizard  Click on: Receptor Timely Integrated Modeller (TIM) Wizard will open in a new window  DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

DockoMatic – TIM Wizard Interface  TIM Wizard DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

TIM Step 1: Enter Template Sequence  Click on: Output Directory DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

TIM Step 1 (cont’d): Sequence Entry  Select: The desired output directory Click: OK  DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

TIM Step 1 (cont’d): Sequence Entry  Output directory path is now established Note this path for future reference  DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

you can copy & paste the sequence here.pdb file OR  Copy/Paste Sequence: If you don’t have a . DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .pdb.TIM Step 1 (cont’d): Sequence Entry  Click on: ‘Sequence’ Choose the target .

TIM Step 1 (cont’d): Sequence Entry

Name the target sequence Click: Next

DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

TIM Step 2: Search for a Template

BLAST Template Search

DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

TIM Step 2 (cont’d): Template List

 

Template list BLAST output

DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

TIM Step 2 (cont’d): Template Selection

Select a template

Templates listed from highest match to lowest
Click: Next

DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

TIM Step 3: Sequence Alignment  Alignment in progress DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

TIM Step 3: Select Aligned Sequence  Select sequence of template that has been aligned with the target DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

Alignment may be edited in this window. Click: Next   DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .TIM Step 4: Edit Aligned Sequence  Aligned template and target sequence display.

TIM Step 5: Model Generation  Model generation in progress DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

TIM Step 5: List of Models Created  List of homology models for target protein created based on template structure DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

TIM Step 5: Select Homology Model Select a homology model Models listed from best to worst correlation Click: Finish    DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

it will be required for molecular docking. Do not close this window.  DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .End TIM Wizard: 3D Model for MD  Receptor field shows selected homology model.

pdb files of the homology models for the target protein along with all other output files generated by DockoMatic. Enter output directory path (you noted this earlier).Location of Model Structure Files • • • • Open a terminal window. DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking . Type: ls The content of the working directory contains the .

Homology Model Validation DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

uk/~rapper/rampage.cam.HM Validation: Ramachandran Plot • Model assessment using RAMPAGE1 webserver DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking 1.ac.php .bioc. http://mordred.

HM Validation: Sequence Alignment DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

12 Å DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .HM Validation: Structure Overlay PDB ID: 2UUR Template Homology model for Col α1 (XI) NPP Root mean square deviation of Cα atoms = 1.

Part II: Grid Parameter File Creation DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

Open AutoDockTools (ADT) • In the terminal type: • adt & • Click on the Icon created at installation DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

then click open.pdbqt file type.pdbqt created from an earlier ADT use. Choose your file. unless loading the .pdb’.• • Click on: Grid Then click ‘Macromolecule’ in the popup menu. • • • DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking . Change the file type to ‘.pdb or a . then ‘Open…’ You can either load a .

then click save • DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .pdbqt Choose your location and filename.• The file will load then imidiately ask you to choose a location to save the converted .

It is described by xyz coordinates. The molecule can be rotated by clicking and dragging. • DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .• • Click on: Grid Then click ‘Grid Box…’ in the popup menu This allows you to define the area of interest in the molecule.

• • Click on: Grid Then click ‘Output’ in the popup menu. then click save • DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking . then ‘Save GPF…’ Choose your location and filename.

Part III: Molecular Docking DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

Molecular Docking: Background   Docking : Molecular simulation of a ligand binding to a receptor Output: Binding energies & scoring functions   Visualization: Binding poses for ligands Docking tools  AutoDock  DOCK  Glide  MOE  GOLD DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

j (Si * Vj * exp ( -rij2 / (2 * σ2) ) ) ΔG = ΔGvdW + ΔGH-bond + ΔGelec + ΔGtor + ΔGdesolv DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking http://autodock.Dij / rij10 + Ehbond ) ΔGelec = Welec ∑i.edu/resources/science/equations/ .Bij / rij6 ) ΔGH-bond = WH-bond ∑i. j ( qi * qj ) / ( ε(rij) * rij ) ΔGtor = Wtor Ntor ΔGdesolv = Wdesolv ∑i (C). j E(t) * ( Cij / rij12 .Force Fields for Autodock Energy Function ΔGvdW = WvdW ∑i. j ( Aij / rij12 .scripps.

Molecular Docking: Protocol http://Autodock.edu DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .scripps.

Molecular Docking in DockoMatic  Click: Output Directory DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

MD: Getting Started  Select output directory  Click: OK DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

MD Step 1: Selecting a Ligand  Click: Ligand DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

pdb) Click: OK  DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .MD Step 1 (cont’d): Ligand Selection  Select a ligand structure file (arixtra.

MD Step 2: Receptor Selection  Click: Receptor DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

MD Step 2 (cont’d): Receptor Selection  Select the homology model of Col α1 (XI) NPP from part 1 as the receptor Click: OK  DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

MD Step 3: Receptor Binding Site Selection  Click: Box Coordinates DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

MD Step 3 (cont’d): Receptor Binding Site Selection  Select a box coordinate file  Click: OK DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

MD Step 4: Job Setup  Select number of AutoDock jobs to run: default=100. for this exercise select 10  Click: New Job DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

MD: Job Status  Your job is not yet started Job status displayed here  DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

MD: Starting the Docking Jobs  Left mouse click to select Right click to access menu  DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

MD: Starting the Docking Jobs (cont’d)  Right click initiates popup window Start Delete Analyze View in PyMOl DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

Start Delete Analyze View in PyMOl DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .MD: Starting the Docking Jobs (cont’d)   Click: Start to submit jobs While jobs are running. review input and output files.

use ‘cd’ to change directory • • Starting a job creates dock directories where results are located Type: cd dock_1 DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking • .Review Files in Terminal Window • Return to terminal window and type ‘ls’ to list files Make sure path is correct. if not.

Part IV: Assessment of Docking Results DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

pdbqt DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .Viewing the Contents of a Structure File • • Result output files Type: more arixtra.

Protein Data Base File for Arixtra • • • arixtra.gpf DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .pdbqt contents Type: q Type: more dock.

Viewing the Contents of a Grid Parameter File • • dock.dpf DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .gpf contents Type: more dock.

dpf file Type: more dock.dpf • DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .Viewing the Contents of a Docking Parameter File • View the contents of the dock.

ligand.gpf –l _gridLog.glg & *****You need not to run autogrid job*****  To run an autodock job you would have to type: autodock4 –p _.DockoMatic: MD Required Files   DockoMatic automation requires a receptor.dlg & *****You need not to run autodock job***** DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .dpf –l _dockLog. and grid parameter file as input GPF creation: To run an autogrid job you would type: autogrid4 –p dock.

DockoMatic: Grid Parameter Files • • • Check gridLog file Type: tail -100 _gridlog.glg end of file contents (last 100 lines) This means autogrid completed successfully DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking • .glg _gridlog.

DockoMatic: Job Status in Terminal • • • Check dockLog Type: tail -100 _docklog.dlg _docklog.dlg end of file contents (last 100 lines) • This means autodock completed successfully DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

MD Result Analysis: Cluster Histogram • To check the Cluster Histogram data • • Type: less _docklog.dlg Scroll down a long way until you get to the Clustering Histogram section. DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

Part VV Visualize the Protein-Ligand Complex DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

Viewing MD Results  Left mouse click to select Right click to display the menu  DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

MD Result Analysis: Visualization  Click: View in PyMOL  A 3D model viewer should open in a new window Start Delete Analyze View in PyMOl DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

MD Analysis: Selecting a Result  Select the top ranked result Click: OK  DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

MD Analysis in PyMOL    Open PyMol Click: File. This is the ligand binding pose for the lowest energy docked complex  DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking . then Open This will open the arixtra_rank_1.pdb file.

PyMOL: View Protein Receptor  Select a protein structure file   Click: Open This will provide the structure of the protein receptor molecule DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

Protein Structure Viewed in PyMOL   3D protein structure Homology model of Col α1 (XI) NPP DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

Ribbon Structure of Protein in PyMOL Collagen α1 (XI) NPP domain DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

Protein-Ligand Complex in PyMol Lowest Ki = -6.51 kcal/mol Arixtra DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

PyMol View of Protein-Ligand Interactions DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

Close up View of Interactions between Arixtra and Binding Site Residues DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

Conclusions   DockoMatic Homology model  Model Creation Validation  Structure  Molecular docking  Docking  Result Studies Analysis DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

Questions! & Thank You DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking .

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