Setting up molecular systems

Overview
Creative Commons License: CC-BY Questions:
  • How to get started modelling a protein and a ligand?

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:
Time estimation: 2 hours
Level: Intermediate Intermediate
Supporting Materials:
Published: Jun 3, 2019
Last modification: Nov 9, 2023
License: Tutorial Content is licensed under Creative Commons Attribution 4.0 International License. The GTN Framework is licensed under MIT
purl PURL: https://gxy.io/GTN:T00053
rating Rating: 5.0 (1 recent ratings, 3 all time)
version Revision: 14

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 in Running molecular dynamics simulations using NAMD) requires access to NAMD Galaxy tools, which can be accessed using the Docker container but are currently not available on any public Galaxy server.

Comment: Audience

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

Agenda

In this tutorial, we will cover:

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

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. Open image in new tab

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.

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:

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.

  • 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: Data upload
  1. Create a new history for this tutorial.

    To create a new history simply click the new-history icon at the top of the history panel:

    UI for creating new history

  2. Import the files from the Zenodo link provided.

    https://zenodo.org/record/2600690/files/7cel_modeled.pdb?download=1
    
    • Copy the link location
    • Click galaxy-upload Upload Data at the top of the tool panel

    • Select galaxy-wf-edit Paste/Fetch Data
    • Paste the link(s) into the text field

    • Press Start

    • Close the window

    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:

    1. Go into Data (top panel) then Data libraries
    2. Navigate to the correct folder as indicated by your instructor.
      • On most Galaxies tutorial data will be provided in a folder named GTN - Material –> Topic Name -> Tutorial Name.
    3. Select the desired files
    4. Click on Add to History galaxy-dropdown near the top and select as Datasets from the dropdown menu
    5. In the pop-up window, choose

      • “Select history”: the history you want to import the data to (or create a new one)
    6. Click on Import

  3. Rename the datasets.
  4. Check that the datatype is correct. The file should have the PDB datatype.

    • Click on the galaxy-pencil pencil icon for the dataset to edit its attributes
    • In the central panel, click galaxy-chart-select-data Datatypes tab on the top
    • In the galaxy-chart-select-data Assign Datatype, select pdb from “New type” dropdown
      • Tip: you can start typing the datatype into the field to filter the dropdown menu
    • Click the Save 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

  • 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. Open image in new tab

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: 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. Open image in new tab

Figure 3: The CHARMM-GUI PDB Reader tool

Select both protein and ligand models

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. Open image in new tab

Figure 4: Select both ligand and protein models in CHARMM-GUI

Manipulate the system

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: Generate PDB’ in the bottom right corner.

Snapshot of CHARMM-GUI renaming section. Open image in new tab

Figure 5: Rename the chains in CHARMM-GUI

Download the output

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. Open image in new tab

Figure 6: CHARMM output from CHARMM-GUI

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: Upload files to Galaxy

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

NAMD

Upload the PDB to CHARMM-GUI

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 Solution Builder 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 Solution Builder tool . Open image in new tab

Figure 7: The CHARMM-GUI Solution Builder tool

Select both protein and ligand models

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. Open image in new tab

Figure 8: Select both ligand and protein models in CHARMM-GUI

Manipulate the system

Hands-on: Make necessary modifications

Rename the hetero chain to BGLC and add disulfide bonds.Press ‘Next Step: Generate PDB’ in the bottom right corner.

Snapshot of CHARMM-GUI renaming section. Open image in new tab

Figure 9: Rename the chains in CHARMM-GUI

Set up the waterbox and add ions

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. Open image in new tab

Figure 10: Setting up a waterbox in CHARMM-GUI
Question

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

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: 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 Toukmaji and Board 1996.

Snapshot of CHARMM-GUI FFT section. Open image in new tab

Figure 11: Setting up a FFT in CHARMM-GUI

Download the output

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. Open image in new tab

Figure 12: NAMD output from CHARMM-GUI

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: Upload files to Galaxy

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

  • step3_pbcsetup.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)