Setting up molecular systems

Overview

question Questions
  • How to get started modelling a protein and a ligand?

objectives Objectives
  • learn about the Protein Data Bank

  • learn how to set up up a model protein and ligand system (with CHARMM-GUI)

  • learn how to upload the system to Galaxy

requirements Requirements

time Time estimation: 2 hours

level Level: Intermediate level level level

Supporting Materials

last_modification Last modification: May 21, 2019

comment Audience

This tutorial is intended for those who are new to the computational chemistry tools in Galaxy.

Introduction

In this tutorial, we’ll cover the basics of molecular modelling by setting up a protein in complex with a ligand and uploading the structure to Galaxy. This tutorial will make use of CHARMM-GUI. Please note that the follow-up to this tutorial (located here) requires access to NAMD Galaxy tools, which can be accessed using the Docker container but are currently not available on any public Galaxy server.

Agenda

In this tutorial, we will cover:

  1. Cellulase and cellulose
    1. Get data
  2. Modelling with CHARMM-GUI
    1. CHARMM
    2. NAMD

Cellulase and cellulose

To start we’ll look at the PDB and find the entry for a fungal enzyme that cleaves cellulose. The enzyme is 7CEL, a hydrolase as seen in the figure.

Snapshot of 7CEL pdb with octaose ligand
Figure 1: 7CEL Cellulase with a short chain cellulose (octaose) ligand

In this section we’ll access the PDB, download the correct structure, import it and view in Galaxy.

details Background: What is the PDB (Protein Data Bank) and format?

The Protein Data Bank (PDB) format contains atomic coordinates of biomolecules and provides a standard representation for macromolecular structure data derived from X-ray diffraction and NMR studies. Each structure is stored under a four-letter accession code. For example, the PDB file we will use is assigned the code 7CEL).

More resources:

details Background: Why choose a cellulase?

Using enzymes to break down abundant cellulose into disaccharide units (cellobiose) is a method to optimise the biofuel process. Barnett et al. 2011

More resources:

Get data

The 7CEL PDB does not include a complete 8 unit substrate and some modelling is required. The correctly modelled substrate is provided for this tutorial.

details More details about the modelling done

  • VMD (visualisation software) was used for atomic placement and CHARMM was used for energy minimisation.
  • The PDB structure contains a mutation at position 217 (glumatate to glutamine). Our structure reverses this.
  • The ligand was modelled separately and inserted into the binding site.

hands_on Hands-on: Data upload

  1. Create a new history for this tutorial.

    tip Tip: Creating a new history

    Click the new-history icon at the top of the history panel

    If the new-history is missing:

    1. Click on the galaxy-gear icon (History options) on the top of the history panel
    2. Select the option Create New from the menu
  2. Import the files from the Zenodo link provided.
    https://zenodo.org/record/2600690
    
    • Copy the link location
    • Open the Galaxy Upload Manager (galaxy-upload on the top-right of the tool panel)

    • Select Paste/Fetch Data
    • Paste the link into the text field

    • Press Start

    • Close the window

    By default, Galaxy uses the URL as the name, so rename the files with a more useful name.

    tip Tip: Importing data from a data library

    As an alternative to uploading the data from a URL or your computer, the files may also have been made available from a shared data library:

    • Go into Shared data (top panel) then Data libraries

    • Find the correct folder (ask your instructor)

    • Select the desired files
    • Click on the To History button near the top and select as Datasets from the dropdown menu
    • In the pop-up window, select the history you want to import the files to (or create a new one)
    • Click on Import
  3. Rename the datasets.
  4. Check that the datatype is correct. The file should have the PDB datatype.

    tip Tip: Changing the datatype

    • Click on the galaxy-pencil pencil icon for the dataset to edit its attributes
    • In the central panel, click on the galaxy-chart-select-data Datatypes tab on the top
    • Select datatypes
    • Click the Change datatype button

Modelling with CHARMM-GUI

It is convenient to set up the molecular system outside Galaxy using a tool such as CHARMM-GUI. Alternative methods are possible - see the GROMACS tutorial for an example. Jo et al. 2016

tip Tip: Viewing figures

  • Some of the figures are screenshots and it may be difficult to make out details
  • Right-click on the image and choose ‘Open image in new tab’ to view
  • Zoom in and out as needed to see the content
CHARMM-GUI interface
Figure 2: The CHARMM-GUI interface

Go to the correct section depending on which MD engine you will be using.

CHARMM

Upload the PDB to CHARMM-GUI

Navigate to CHARMM-GUI and use the Input Generator, specifically the PDB Reader tool and upload the Cellulase PDB file. Press ‘Next Step: Select Model/Chain’ in the bottom right corner.

hands_on Hands-on: Upload the PDB to CHARMM-GUI

  1. Retrieve the modelled PDB structure from Zenodo.
  2. Upload the PDB and choose CHARMM format.
Snapshot of CHARMM-GUI PDB reader section
Figure 3: The CHARMM-GUI PDB Reader tool

Select both protein and ligand models

hands_on Hands-on: Generate PDB file

Two model chains are presented for selection: the protein (PROA) and the hetero residue, which is the ligand or glycan in this case (HETA). Select both, and press ‘Next Step: Generate PDB’ in the bottom right corner.

Snapshot of CHARMM-GUI model section
Figure 4: Select both ligand and protein models in CHARMM-GUI

Manipulate the system

hands_on Hands-on: Make necessary modifications

Rename the hetero chain to BGLC and add ten disulfide bonds to the protein, as shown in the figure. Then press ‘Next Step: Manipulate PDB’ in the bottom right corner.

Snapshot of CHARMM-GUI renaming section
Figure 5: Rename the chains in CHARMM-GUI

Download the output

hands_on Hands-on: Download CHARMM output

The output is a .tgz file (a tarball or zipped tarball). Inside the archive you will see all inputs and outputs from CHARMM-GUI.

Snapshot of CHARMM-GUI CHARMM output section
Figure 6: CHARMM output from CHARMM-GUI

tip What is a .tgz file?

This is a compressed file which contains all the output files created by the CHARMM-GUI. To access them, the .tgz file needs to be decompressed. There should be a tool available on your operating system for this. If you prefer to use the command line, tar will work fine on Linux or Mac tar -zxvf example.tgz. On Windows use 7zip, or download Git for windows and use Git Bash.

Upload to Galaxy

hands_on Hands-on: Upload files to Galaxy

Upload the step1_pdbreader.psf and step1_pdbreader.crd files to your BRIDGE instance and run the system setup tool.

NAMD

Upload the PDB to CHARMM-GUI

hands_on Hands-on: Upload the PDB to CHARMM-GUI

Retrieve the modelled PDB structure from Zenodo. Navigate to CHARMM-GUI and use the Input Generator, specifically the Quick MD Simulator tool. Upload the PDB file, selecting ‘CHARMM’ as the file format. Press ‘Next Step: Select Model/Chain’ in the bottom right corner.

Snapshot of CHARMM-GUI Quick MD Simulator tool
Figure 7: The CHARMM-GUI Quick MD Simulator tool

Select both protein and ligand models

hands_on Hands-on: Generate PDB file

Two model chains are presented for selection: the protein (PROA) and the hetero residue, which is the ligand or glycan in this case (HETA). Select both, and press ‘Next Step: Generate PDB’ in the bottom right corner.

Snapshot of CHARMM-GUI model section
Figure 8: Select both ligand and protein models in CHARMM-GUI

Manipulate the system

hands_on Hands-on: Make necessary modifications

Rename the hetero chain to BGLC and add disulfide bonds.

Snapshot of CHARMM-GUI renaming section
Figure 9: Rename the chains in CHARMM-GUI

Set up the waterbox and add ions

hands_on Hands-on: Solvate the protein

Set up a waterbox. Use a size of 10 angstroms and choose a cubic box (‘rectangular’ option).

Snapshot of CHARMM-GUI waterbox section
Figure 10: Setting up a waterbox in CHARMM-GUI

question Question

Why is 10 angstrom a fair choice for the buffer? Why choose 0.15M NaCl?

solution Solution

Under periodic boundary conditions, we need to ensure the protein can never interact with its periodic image, otherwise artefacts are introduced. Allowing 10 angstroms between the protein and the box edge ensures the two images will always be at minimum 20 angstroms apart, which is sufficient.

Some of the residues on the protein surface are charged and counter-ions need to be present nearby to neutralise them. Failure to explicitly model salt ions may destabilise the protein.

Generate the FFT automatically

hands_on Hands-on: Generate the FFT

Particle Mesh Ewald (PME) summation is the method being used to calculate long-range interactions in this system. To improve the computational time a Fast Fourier Transform (FFT) is used. A detailed discussion of FFT will not be presented here; there are many articles on the subject. Try Wikipedia and Ewald summation techniques in perspective: a survey.

Snapshot of CHARMM-GUI FFT section
Figure 11: Setting up a FFT in CHARMM-GUI

Download the output

hands_on Hands-on: Solvate the protein

The output is a .tgz file (a tarball or zipped tarball). Inside the archive you will see all inputs and outputs from CHARMM-GUI.

Snapshot of CHARMM-GUI NAMD output section
Figure 12: NAMD output from CHARMM-GUI

tip What is a .tgz file?

This is a compressed file and needs to be uncompressed using the correct tool. On Linux or Mac: tar will work fine tar -zxvf example.tgz. On Windows use 7zip or download Git for windows and use Git Bash.

Upload to Galaxy

hands_on Hands-on: Upload files to Galaxy

Upload the following files to your BRIDGE instance and ensure the correct datatype is selected:

  • step3_pbcsetup.xplor.ext.psf -> xplor psf input (psf format)
  • step3_pbcsetup.pdb -> pdb input (pdb format)
  • Checkfft.str -> PME grid specs (txt format)
  • step2.1_waterbox.prm -> waterbox prm input (txt format)

You are now ready to run the NAMD workflow, which is discussed in another tutorial.

Conclusion

trophy Well done! You have started modelling a cellulase protein and uploaded it into Galaxy. The next step is running molecular dynamics simulations (tutorial)

keypoints Key points

  • The PDB is a key resource for finding protein structures.

  • Using CHARMM-GUI is one way to prepare a protein and ligand system.

  • To get data into Galaxy you can upload a file from your computer or paste in a web address.

Useful literature

Further information, including links to documentation and original publications, regarding the tools, analysis techniques and the interpretation of results described in this tutorial can be found here.

References

  1. Barnett, C. B., K. A. Wilkinson, and K. J. Naidoo, 2011 Molecular Details from Computational Reaction Dynamics for the Cellobiohydrolase I Glycosylation Reaction. Journal of the American Chemical Society 133: 19474–19482. 10.1021/ja206842j
  2. Jo, S., X. Cheng, J. Lee, S. Kim, S.-J. Park et al., 2016 CHARMM-GUI 10 years for biomolecular modeling and simulation. Journal of Computational Chemistry 38: 1114–1124. 10.1002/jcc.24660

congratulations Congratulations on successfully completing this tutorial!



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