dockomatic : automated tool for homology modeling and docking studies

DockoMatic:Automated Tool for Homology Modeling and Docking Studies

DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Tutorial

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


Systems of Interest Protein: Collagen α1 (XI) NPP domain

223 amino acid protein 2 disulfide bonds A minor fibrillar collagen involved in

thrombosis

Drug: Arixtra (a.k.a. Fondaparinux) A heparin-based drug An anticoagulant used to treat blood clots Binds Col α1 (XI) NPP to prevent blood

coagulation O

OO

OO

-O3SO

OH

OH

NHSO3-

O

O O

OH

OH

O

-O3SO

OSO3-

NHOSO3-

O

COO-OH

OSO3-

O

OH

-O3SO

O CH3

NHSO3-

Arixtra structure


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.

How many structures solved? The Protein Data

Bank*Dposition Year X-Ray Structures Total X-Ray Structures1995 971 9711996 1148 21191997 1503 36221998 1793 54151999 2149 75642000 2457 100212001 2657 126782002 2782 154602003 3917 193772004 4454 238312005 5131 289622006 5664 346262007 6695 413212008 6115 474362009 7357 547932010 7790 625832011 8308 708912012 3953 74844

*Bernstein, et al. (1977) J. Mol. Biol. 112, 535.


Part I: Homology Modeling


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, activity, ligand binding paradigms in drug design, etc.


Homology Model: Template Selection

1. Begin with the target amino acid sequence

2. Use the Basic Local Alignment Search Tool* (BLAST) to search for amino acid sequences of template proteins where the 3D structure is available

3. The principle repository for 3D protein structures is the National Center for Biotechnology Information+ (NCBI)

4. Only amino acid sequences with greater than 30% identity between target and template should be considered.

5. If functionality is known for a class or family of proteins, look to protein templates with similar functionality as the target.


*Johnson et al. (2008) Nucleic Acids Res. 36, W5-9. +Edgar, et al. (2002) Nucleic Acids Res. 30, 207-210

Homology Modeling: Flow Chart

Target Sequence

Model Building for the Target

Template Selection

Alignment (Target-

Template)

Evaluate the

Model

Known (Template) Structures

Homology Model

YESNO


Homology Model Creation:

DockoMatic Procedure


• Open Terminal Window


• Type: ssh –X username@host


• Enter password


• This is the HOME directory


• Type: cd $SCRATCH


• Type: mkdir DOCKOMATIC-WORKSHOP

• Type: cd DOCKOMATIC-WORKSHOP

• Type: dockomatic

• DockoMatic will now open in a new window


DockoMatic 2.0 GUI


DockoMatic

Select:

Use Modeller


DockoMatic: Accessing the TIM Wizard

Click on:

Receptor Timely

Integrated Modeller (TIM) Wizard will open in a new window


DockoMatic – TIM Wizard Interface

TIM Wizard


TIM Step 1: Enter Template Sequence

Click on:

Output Directory


TIM Step 1 (cont’d): Sequence Entry

Select: The desired output directory

Click: OK


Output directory path is now established

Note this path for future reference



Click on:

‘Sequence’

Choose the target .pdb file

OR

Copy/Paste Sequence:

If you don’t have a .pdb, you can copy & paste the sequence here.



Name the target sequence

Click: Next



TIM Step 2: Search for a Template

BLAST Template Search


Template list BLAST

output


TIM Step 2 (cont’d): Template List

Select a template

Templates listed from highest match to lowest

Click: Next

TIM Step 2 (cont’d): Template Selection


TIM Step 3: Sequence Alignment

Alignment in progress


Select sequence of template that has been aligned with the target

TIM Step 3: Select Aligned Sequence


Aligned template and target sequence display.

Alignment may be edited in this window.

Click: Next

TIM Step 4: Edit Aligned Sequence


TIM Step 5: Model Generation

Model generation in progress


List of homology models for target protein created based on template structure


TIM Step 5: List of Models Created

Select a homology model

Models listed from best to worst correlation

Click: Finish

TIM Step 5: Select Homology Model


Receptor field shows selected homology model.

Do not close this window; it will be required for molecular docking.

End TIM Wizard: 3D Model for MD


• Open a terminal window.

• Enter output directory path (you noted this earlier).

• Type: ls

• The content of the working directory contains the .pdb files of the homology models for the target protein along with all other output files generated by DockoMatic.

Location of Model Structure Files


Homology Model Validation


HM Validation: Ramachandran Plot• Model assessment using

RAMPAGE1 webserver

DOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking1. http://mordred.bioc.cam.ac.uk/~rapper/rampage.php

HM Validation: Sequence Alignment


HM Validation: Structure Overlay

Homology modelfor Col α1 (XI) NPP

PDB ID: 2UUR Template

Root mean square deviation of Cα atoms = 1.12 Å


Part II: Grid Parameter File Creation


Open AutoDockTools

(ADT)• In the terminal type:

• adt &

• Click on the Icon created at installation


• Click on: Grid

• Then click ‘Macromolecule’ in the popup menu, then ‘Open…’

• You can either load a .pdb or a .pdbqt created from an earlier ADT use.

• Change the file type to ‘.pdb’, unless loading the .pdbqt file type.

• Choose your file, then click open.



• The file will load then imidiately ask you to choose a location to save the converted .pdbqt

• Choose your location and filename, then click save


• Click on: Grid

• Then click ‘Grid Box…’ in the popup menu

• This allows you to define the area of interest in the molecule. It is described by xyz coordinates. The molecule can be rotated by clicking and dragging.


• Click on: Grid

• Then click ‘Output’ in the popup menu, then ‘Save GPF…’

• Choose your location and filename, then click save

Part III: 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 GOLDDOCKOMATIC-Student Procedure-Homology Modeling & Molecular Docking

Force Fields for Autodock Energy Function

http://autodock.scripps.edu/resources/science/equations/

ΔGH-bond = WH-bond ∑i, j E(t) * ( Cij / rij12 - Dij / rij

10 + Ehbond )

ΔGvdW = WvdW ∑i, j ( Aij / rij12 - Bij / rij

6 )

ΔGelec = Welec ∑i, j ( qi * qj ) / ( ε(rij) * rij )

ΔGdesolv = Wdesolv ∑i (C), j (Si * Vj * exp ( -rij2 / (2 * σ2) ) )

ΔGtor = Wtor Ntor

ΔG = ΔGvdW + ΔGH-bond + ΔGelec + ΔGtor +

ΔGdesolv


Molecular Docking: Protocol

http://Autodock.scripps.edu


Click: Output Directory

Molecular Docking in DockoMatic


Select output directory

Click: OK

MD: Getting Started


Click: Ligand

MD Step 1: Selecting a Ligand


Select a ligand structure file (arixtra.pdb)

Click: OK

MD Step 1 (cont’d): Ligand Selection


Click: Receptor

MD Step 2: Receptor Selection


Select the homology model of Col α1 (XI) NPP from part 1 as the receptor

Click: OK

MD Step 2 (cont’d): Receptor Selection


Click: Box Coordinates

MD Step 3: Receptor Binding Site Selection


Select a box coordinate file

Click: OK


MD Step 3 (cont’d): Receptor BindingSite Selection

Select number of AutoDock jobs to run: default=100; for this exercise select 10

Click: New Job

MD Step 4: Job Setup


Your job is not yet started

Job status displayed here

MD: Job Status


Left mouse click to select

Right click to access menu


MD: Starting the Docking Jobs

StartDeleteAnalyzeView in PyMOl

Right click initiates pop-up window

MD: Starting the Docking Jobs (cont’d)



Click: Start to submit jobs

While jobs are running, review input and output files.


MD: Starting the Docking Jobs (cont’d)

• Return to terminal window and type ‘ls’ to list files

• Make sure path is correct; if not, use ‘cd’ to change directory

• Starting a job creates dock directories where results are located

• Type: cd dock_1


Review Files in Terminal Window

Part IV: Assessment of Docking Results


• Result output files

• Type: more arixtra.pdbqt


Viewing the Contents of a Structure File

• arixtra.pdbqt contents

• Type: q

• Type: more dock.gpf


Protein Data Base File for Arixtra

• dock.gpf contents

• Type: more dock.dpf


Viewing the Contents of a Grid Parameter File

• View the contents of the dock.dpf file

• Type: more dock.dpf

Viewing the Contents of a Docking Parameter File


DockoMatic: MD Required Files

DockoMatic automation requires a receptor, ligand, and grid parameter file as input

GPF creation: To run an autogrid job you would type:

autogrid4 –p dock.gpf –l _gridLog.glg &

*****You need not to run autogrid job*****

To run an autodock job you would have to type:

autodock4 –p _.dpf –l _dockLog.dlg &

*****You need not to run autodock job*****


• Check gridLog file

• Type: tail -100 _gridlog.glg

• _gridlog.glg end of file contents (last 100 lines)

• This means autogrid completed successfully

DockoMatic: Grid Parameter Files


• Check dockLog

• Type: tail -100 _docklog.dlg

• _docklog.dlg end of file contents (last 100 lines)

• This means autodock completed successfully


DockoMatic: Job Status in Terminal

• To check the Cluster Histogram data

• Type: less _docklog.dlg

• Scroll down a long way until you get to the Clustering Histogram section.


MD Result Analysis: Cluster Histogram

Part VV Visualize the Protein-Ligand Complex


Left mouse click to select

Right click to display the menu

Viewing MD Results



Click:

View in PyMOL

A 3D model viewer should open in a new window

MD Result Analysis: Visualization


Select the top ranked result

Click: OK

MD Analysis: Selecting a Result


Open PyMol Click: File, then

Open This will open the

arixtra_rank_1.pdb file.

This is the ligand binding pose for the lowest energy docked complex

MD Analysis in PyMOL


Select a protein structure file

Click: Open This will

provide the structure of the protein receptor molecule

PyMOL: View Protein Receptor


3D protein structure

Homology model of Col α1 (XI) NPP

Protein Structure Viewed in PyMOL


Collagen α1 (XI) NPP domain


Ribbon Structure of Protein in PyMOL

Protein-Ligand Complex in PyMol

Arixtra

Lowest Ki = -6.51 kcal/mol


PyMol View of Protein-Ligand Interactions


Close up View of Interactions between Arixtra and Binding

Site Residues


Conclusions

DockoMatic Homology model

Model Creation Structure Validation

Molecular docking Docking Studies Result Analysis


Questions!&

Thank You


dockomatic : automated tool for homology modeling and docking studies

Documents

protein homology model

protein templates

d protein structures

given protein sequence

target amino acid sequenceuse

protein data bank

template selectionbegin

useful model