View source for Gromacs/C3/Production-Run-for-Lysozyme/English


{| border=1
|  | '''Visual Cue'''
|  | '''Narration'''

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|| '''Slide Number 1'''

'''Title Slide '''
|| Welcome to the spoken tutorial on '''Production Run for Lysozyme'''.
|-
|| '''Slide Number 2'''

'''Learning Objectives'''
|| In this tutorial, we will,
* Complete 1 nanosecond production run for '''lysozyme'''
* About timescales of motions
* Time taken for simulation
* Load the '''xtc''' file for trajectory
* Calculate '''RMSD''' from trajectory

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|| '''Slide Number 3'''

'''Learning Objectives'''
|| 
* Save data to various output files
* Align a frame in the trajectory with a reference molecule

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|| '''Slide Number 4'''

'''System and Software Requirement'''
|| To record this tutorial, I am using

* '''Ubuntu Linux''' v20.04 OS
* '''Gromacs''' v2021.2
* '''VMD''' 1.9.3

|-
|| '''Slide Number 5'''

'''Pre-requisites'''

[https://spoken-tutorial.org/ https://spoken-tutorial.org] '''
|| To follow this tutorial, 
* Learner must be familiar with basics of '''Gromacs''' and '''VMD'''.
* For pre-requisite tutorials, please visit this site.

|-
|| '''Slide Number 6'''

'''Code Files'''
|| 
* Files used in this tutorial are provided in the '''code files''' link.
* Please download and extract the files.
* Make a copy and then use them while practising.

|-
|| Show input files.

Show, copied, files in '''firsmd''' directory.
|| Several files are provided with this tutorial.


First, copy, the '''mdp''' file to the working directory.

If needed, copy the required files from the previous step.

|-
|| Open '''Ubuntu 20.04 LTS app''' ('''Windows''') or '''terminal''' ('''Linux''').

Type '''cd Documents/firstmd''' and press '''Enter'''.
|| Open a terminal and go to the working directory.

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|| Type '''cd ls''' and press '''Enter'''.
|| I will also list the files.

|-
|| Cursor on '''npt.gro''' and '''npt.tpr'''.
|| The structure and configuration files for '''MD''' simulation are '''npt.gro''' & '''npt.tpr'''. 

At this point, the following has happened to the starting '''PDB''' file.

|-
|| Show all the files in the folder.
|| The protein is solvated, charge neutralized with ions and energy minimized.

Then, in the initial MD step, temperature and pressure was equilibrated.

This prepared the system for the Production run MD.

I have all the files from all the steps in this folder.

|-
|| Cursor on '''md.mdp''' and open in a text editor.
|| Open the '''mdp''' file provided for '''MD''' simulation in a text editor.

|-
|| Cursor on the time.
|| Here, the timescale of simulation is 1 '''ns'''.

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|| 
|| This allows us to follow changes in the conformation of the protein.

In energy minimization and equilibration steps, the time duration was shorter.

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|| Back to the text editor and close the '''mdp''' file.
|| Often, step size and length of simulation are adjusted.

This file specifies ½ million steps in 1 ns.

Frequency with which structures are written to the trajectory is also adjustable.

Let’s close the '''mdp''' file.

|-
|| '''Slide Number 7'''

'''Timescale of Protein Motions'''


[[Image:timescale.png|top|500px]]
|| This image shows the timescale of molecular motions we usually encounter.

The ns-ps time encompasses the lower end of protein motions for classical MD.

|-
|| '''Slide Number 8'''

'''Timescale of Protein Motions'''
|| 
* In microsecond or millisecond timescale, ligand binding or folding is studied.
* These are computationally intensive processes
* Use a high performance computation facility or a '''GPU''' for longer calculations

|-
|| '''Slide Number 9'''

'''Timescale of Protein Motions'''
|| 
* Use a high performance computation facility or a '''GPU''' for longer calculations

|-
|| Go to the '''terminal'''.
|| Go to the '''terminal'''.

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|| Type, '''gmx grompp -f md.mdp -c npt.gro -t npt.cpt -p topol.top -o md-1.tpr''' and press '''Enter'''.
|| Enter the command as seen here to assemble the '''configuration''' for the '''md''' run.

'''npt.gro is''' the starting structure for the '''production md run'''.

|-
|| Cursor on '''md-1.tpr'''.
|| The '''tpr''' file is the output file from this step.

An '''r''' flag is used if we need to restrain the protein conformation.

This is often done when studying protein-ligand binding.

Here we will not used it.

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|| Cursor on the message.
|| While creating the file, few messages are seen on the '''terminal'''.

|-
|| Type '''ls''' and press '''Enter'''.
|| Enter '''ls''' on the '''terminal''' to list the files. 

Notice the output '''md-1.tpr''', that is created.

This prepared the system for the '''production md'''.

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|| Press '''Ctrl+L''' to clear the screen.
|| As we did for the energy minimization step, we will enter the next command.

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|| Type, '''gmx mdrun -v -deffnm md-1''' and press '''Enter'''.
|| Enter the '''mdrun''' command as seen to start the '''production run'''.

Usually, molecular dynamics refers to equilibration followed by production run.

|-
|| Show the output on the '''terminal'''. 
|| This time, the '''verbose flag''' shows only a few details. 

The number of steps and time needed to complete are seen on the screen.

|-
|| Show the time taken for the process.
|| Process will stop when it reaches 500 thousand steps in 1 ns.

This process may take a day or so on many personal computers.

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|| Press '''Ctrl+C'''.
|| You may run this process later on.

Use the provided data for further analysis.

To abort the process, press '''Control''' and '''C''' keys together once.

In a few minutes '''Gromacs''' stops the process.

|-
|| Show files in the file manager.
|| Several files with '''md''' prefix are generated in the working directory.

|-
|| Highlight and delete the files to be deleted.
|| If you had aborted the process, delete the incomplete files that are created.

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|| 
|| If you have completed the process, do not delete the files.

A '''gro''' file will get created at the completion of the process.

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|| Show files to be copied from provided file. 

Copy them to the working directory.
|| Copy the provided files with the '''md-1''' prefix to the working directory.

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|| Type '''ls -ltr''' and press '''Enter'''.
|| This step creates additional file types with '''cpt''' and '''xtc''' extensions.
|-
|| Cursor on '''md-1.xtc''' file.
|| The '''xtc''' file is a compressed trajectory file.


This file is useful, in data analysis during long simulations.
|-
|| Cursor on '''md.trr''', '''md.log''' and '''md.edr''' files.
|| '''Trr''', '''log''' and '''edr''' files are also created in this process.
|-
|| Show the output files.
|| In case of errors, learner may refer to the '''log''' file for trouble shooting.

|-
|| 
|| We have generated several files for further analysis.

Data analysis path varies depending on our aim or objective of simulation.

|-
|| '''Slide Number 9'''

'''Data Analysis'''
|| Few examples of data analysis are,
* Validation, visualization
* Analysis of energetics, H-bonding
* Secondary structure changes
* Ligand Binding, surface analysis, 
* Analysis of subset of atoms in the system

|-
|| Open a web browser.

Go to, [https://manual.gromacs.org/documentation/2018/user-guide/cmdline.html https://manual.gromacs.org/documentation/2018/user-guide/cmdline.html] .
|| Open a web browser.

Let’s go to the '''Gromacs''' manual site as seen here.

|-
|| Cursor on the left panel.
|| On the left frame notice the various categories of topics for analysis.

Atomic interactions, kinetics and '''PCA''' are also options.

These are useful to analyze flexible structures or molten globules.

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|| 
|| Learner is encouraged to read further on this to know more.

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|| Go to the '''terminal'''.

Press '''Ctrl+L'''.
|| Go to the terminal.

I will clear the '''terminal''' for clarity in the video.

|-
|| Type, '''vmd md-1.gro md-1.xtc''' and press '''Enter'''.
|| Let’s open '''VMD''' to view the trajectory from the 1'''ns''' long simulation.

This time we will use the '''xtc''' file instead of the '''trr''' file to load the '''trajectory'''.

'''Xtc''' is a compressed format of the simulation trajectory.

'''Windows''' users must first open '''VMD'''.

Then open the '''gro''' file, followed by the '''xtc '''trajectory''' file.

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|| Click on '''Graphics, representation'''.


Type, '''protein''' for '''Selected atoms''' and press '''Enter'''.
|| Hiding the water molecules from graphics display will be useful for clarity.

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|| 
|| You may adjust the graphic representation of the protein to your desire.

I will change it to show as ribbons.

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|| In the '''VMD''' terminal enter the command '''pbc box'''.
|| Display the box, using the command as seen here in the '''vmd terminal'''.

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|| Play the trajectory.
|| Pause the video and play the trajectory.

Notice the '''xtc''' file has 1002 frames.

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|| 
|| Later on, you may load the '''trr''' file and notice, it has only 101 frames.

'''Xtc''' format has precision of 3 decimal places compared to 6 decimals for '''trr'''.

Hence, even though '''xtc''' has 10 times more frames, the file sizes are comparable.

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|| Stop playing the trajectory.

Close the Window.
|| Visualization alone is not sufficient to know the features of the data.

Hence let’s do few data analysis to see the features embedded.

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|| Click on '''Extensions, Analysis''' and choose '''RMSD Trajectory Tool'''.
|| Click on '''Extensions, Analysis''' and choose '''RMSD Trajectory Tool'''.

A '''RMSD trajectory tool''' window opens.

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|| 
|| Let’s calculate '''RMSD''' of the backbone atoms in the trajectory.

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|| In '''Selection modifiers''', check the box for '''Backbone'''.
|| In '''Selection modifiers''', check the box for '''Backbone'''.

Notice that, '''Trace''' or '''NH''' can also be selected for plotting. 

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|| Cursor over trajectory section.
|| From the '''Trajectory''' section, we can also select a range of frames for analysis.

I will retain the defaults and check the boxes for '''Plot''' and '''Save'''.

|-
|| Cursor on '''trajrmsd.dat'''.
|| Notice that, the data will also get saved with the given filename.

You may change it if you desire.

I will leave it as is.

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|| Click on '''md-1.gro''' in '''mol''' section.
|| In the molecule section, click on '''md-1.gro''' file to select it.

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|| Click on the '''RMSD'''.

Show the graph.
|| Click on the '''RMSD''' button and the graph is seen.

'''X''' axis is the frame number and '''y''' axis is the '''RMSD''' in the graph.

|-
|| Click on '''File''' in graphics window.
|| We can also export the data in many different formats as seen here. 

|-
|| Screenshot of the file manager and output file.
|| The data can also be written to a text file in two column format.

Learner may use a plotting program of your choice to plot it.

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|| Close the '''RMSD Trajectory Tool''' window.
|| Let’s close this window.

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|| Cursor on the graphics window.
|| Now, let’s display frame 50 and align it to '''md-1.gro'''.

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|| Close the '''RMSD''' graphical window.

Go to '''representations''' and click on '''Create Rep'''.
|| Go to '''representations''' window and create a new representation.

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|| Go to the '''Trajectory''' tab.
|| Go to the '''Trajectory''' tab.

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|| Cursor on '''Draw multiple frames '''form, type, 50 and press '''Enter'''.
|| In the '''Draw multiple frames''' form, type 50 and press '''Enter'''.

This shows the 50th frame also.

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|| Open the '''RMSD Trajectory Tool'''.
|| To align them, go back to the '''RMSD Trajectory Tool'''.

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|| For '''Reference mol''', choose '''Selected'''.
|| For '''Reference mol''', choose '''Selected'''.

|-
|| Type 50 for '''Reference frame'''.
|| Type 50 for '''Reference frame'''.

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|| Uncheck the the box for '''Plot'''.
|| I will uncheck the box for '''Plot'''.

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|| Click on '''md-1.gro''' in '''mol''' section.
|| In the molecule section, click on '''md-1.gro''' file to select it.

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|| Click on '''ALIGN'''.

Show alignment in the graphical window.
|| Now, click on '''ALIGN''' and notice the two aligned molecules.

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|| Click on '''FIle, Save Visualization State'''.

In the '''filename''' form, type filename '''analysis.vmd''' and click on '''Ok'''.
|| Click on '''FIle, Save Visualization State''' to save the state in '''VMD'''.

Enter file name '''analysis.vmd''' and retain the working directory to save the file.

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|| Show the '''Options''' menu.
|| Pause the video and explore the '''Options''' menu.

Various file formats and options are available for user convenience.

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|| Screenshot of '''energy''' command options to export to '''xvg''' file.
|| You may further, extract and plot more parameters.

|-
|| '''Slide Number 10'''

'''Summary '''
|| Now let’s summarize. In this tutorial, we,

* Completed 1ns Production run for '''lysozyme'''
* About timescales of motions
* Time taken for simulation
* About output data and various types of data analysis

|-
|| '''Slide Number 11'''

'''Summary'''
|| * Loaded the '''xtc''' trajectory file
* About '''RMSD trajectory tool''' and analysis
* Aligned a frame in the trajectory with a reference molecule

|-
|| '''Slide Number 12'''

'''Assignment'''
|| For assignment activity, please do the following. 

* Open the '''log''' file and go through the details
* Plot '''RMSD''' of '''trace''' (C-alpha) atoms
* Create '''Ramachandran''' plot of the lowest and highest energy structure

|-
|| '''Slide Number 13'''

'''Assignment'''
|| Using the '''energy '''command, 

* Plot '''solvent accessible surface area''' ('''SASA''')
* Explore '''gmxcheck''', and '''ngmx''' commands

|-
|| '''Slide Number 14'''

'''Spoken Tutorial Project'''
|| This video summarises the Spoken Tutorial Project.

Please download and watch it.

|-
|| '''Slide Number 15'''

'''Spoken Tutorial workshops'''
|| We conduct workshops using spoken tutorials and give certificates. 

Please write to us.

|-
|| '''Slide Number 15'''

'''Forum for questions'''
|| Post your timed queries in this forum.

|-
|| '''Slide Number 16'''

'''Acknowledgment'''
|| Spoken Tutorial Project is funded by MoE, Government of India.

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|| 
|| This is Rani from IIT, Bombay. Thank you for joining.

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|}