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Introduction

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Getting started with Linux
Prepare topology and coordinate files
Load a protein and nucleic acid force field
Build alanine dipeptide
Solvate alanine dipeptide
Save the Amber parm7 and rst7 input files
Prepare Amber MD sander input files
Minimization input
Heating input
Production input
Run Amber MD sander
Run minimization
Run heating MD
Run production MD
Visualize the results
Analyze the MD results
Production MD system properties
Use cpptraj to analyze the RMSD
Conclusion
Appendix: tutorial files
Reference and links
This tutorial is designed to provide an introduction to molecular dynamics simulations with Amber. It is designed around AMBER Tools v14 and assumes that you have not used Linux or Amber before. It is designed for new users who want to learn about how to run Molecular Dynamics simulations. It does however assume that you have a machine with AmberTools v15, VMD and xmgrace correctly installed.
AMBER stands for Assisted Model Building and Energy Refinement. It refers not only to the molecular dynamics programs, but also a set of force fields that describe the potential energy function and parameters of the interactions of biomolecules.
In order to run a Molecular Dynamics simulation in Amber, each molecule's interactions are described by a molecular force field. The force field has specific parameters defined for each molecule.
sander is the basic MD engine of Amber. pmemd is the high performance implementation of the MD engine that contains a subset of features of sander. pmemd can also be run with acceleration from graphics processing units (GPU).
In order to run an MD simulation with sander or pmemd, three key files are needed:

1. parm7 - The file that describes the parameter and topology of the molecules in the system
2. rst7 - The file that describes the initial molecular coordinates of the system
3. mdin - The file that describes the settings for the Amber MD engine

Amber MD is software that is entirely based on a Command Line Interface (CLI) on a computer with Linux. To run Amber, you will need to open a terminal.
1. Open a terminal now on your Linux computer.
On most Linux machines, your terminal will look like this:

Most of the work for this tutorial will be done mainly through the terminal.

List files and make a directory (folder) to store your files in

Whenyou first log in and start a terminal, your current working directory (or folder) is your home directory. It has the same name as your username, and it is where your files and directories are stored. In most cases, this is /home/username

ls (list)

2. Use the ls command to list what is in your current directory.
Note that the '$' is the command line prompt in the terminal. At this point there will probably be some files and directories in your home directory that are created automatically with your account.

mkdir (make directory)

You'll need a new directory for the files and folders created in this tutorial.

3. Make a new directory for this tutorial with the mkdir command called Tutorial.

Now when you do ls you should see your new directory has been created.

cd (change directory)

At this point, you'll want to move into your Tutorial directory so that you can save all of your working files there.

4. Use the cd command to change to different directory.

There is a special directory named '.'. This means the parent of the current directory. So to return to the parent of the Tutorial directory use cd . If you ever need to return to your home directory use just the cd command by itself. Tilde '~' is a shortcut to your home directory. The following commands both change directory to your home directory.

pwd (print working directory)

Pathnames describe what directory you are in relative to the entire computer's filesystem. You home directory has location within the entire filesystem.
5. Print the working directory pathname of your home directory with pwd. This is the current working directory that you're located in. In this case the directory username is in the directory home which is in the / (root) directory. For this tutorial, you will build the following molecule in the preparatory program called xLEaP for simulation in AMBER. In order to build and solvate this molecule, you will need to start xLEaP. xLEaP has another command line interface and simple molecular graphics for building the system topology and define parameters for the molecules.
6. Start xLEaP now with the xleap command. You should see a window like this:
Warning:
Do NOT click the 'X' on any LEaP window. It will quit LEaP entirely.
Note:
At this point it's a good idea to turn Num Lock off for the menus to work.

Load a protein and nucleic acid force field

A MD force field is the the Hamiltonian (potential energy function) and the related parameters that describe the intra- and intermolecular interactions between the molecules in the system. In MD, the Hamiltonian is integrated to describe the forces and velocities of the molecules.
The basic form of the Amber Hamiltonian is:

In order to run a molecular dynamics simulation, we need to load a force field to describe the potential energy of alanine dipeptide. We will use the AMBER force field FF14SB for proteins and nucleic acids. FF14SB is based off FF12SB, an updated version of FF99SB, which in turn was based on the original Amber Cornell et al (1995) [ff94] force field. The most significant changes to the FF14SB force field included updated torsion terms for the protein Phi-Psi angles and refits of the torsion terms for side chains. Together these improved the estimation of alpha helices in these molecules.
7. source (load) the FF14SB force field now.

Build alanine dipeptide

We can build an alanine dipeptide as an alanine amino acid capped with an acetyl group on the N-terminus and n-methylamide on the C-terminus. After we loaded the force field ff14SB, xLEaP now has these 'units' available to build into a molecule. The sequence command will create a new unit from the available units and connect them together.
8. Use sequence to create a new unit called foo out of theACE, ALA, and NME units.
Note that the '>' indicates the command line in xLEaP. Now you have a single alanine dipeptide molecule stored in the unit foo. xLEaP provides a very basic editor to examine and change units and molecules.
9. Examine the structure of the alanine dipeptide molecule. Use the edit command to view the structure. The editing window will look like this:
From here, you can examine the topology, structure, atom names, atom types, and partial charges of the molecule. Basic editing of the molecule is also possible.
Warning:
Do NOT click the 'X' to close this window. It will exit xLEaP. To close this window, use Unit -> Close.

Solvate alanine dipeptide

The next step to prepare this alanine dipeptide system is to solvate the molecule with explicit water molecules. In this simulation we will use add TIP3P water molecules to the system.

In this type of simulation, the system has periodic boundary conditions, meaning that molecules that exit one side of the system will wrap to the other side of the system. It is important that the periodic box is large enough, i.e. there is enough water surrounding alanine dipeptide, so that the alanine dipeptide molecule does not interact with its periodic images.

There are many water models available for MD simulations. Fashion design sketches male. However, for this tutorial we will use the TIP3P water model for this simulation.

10. Solvate the system with the solvatebox command.

TIP3PBOX specifies the type of water box to solvate with. 10.0 indicates that the molecule should have a buffer of at least 10 Angstroms between alanine dipeptide and the periodic box wall.

Save the Amber parm7 and rst7 input files

Now we will save the parm7 and rst7 files to the current working directory. The unit foo now contains the alanine dipeptide molecule, water molecules, and the periodic box information necessary for simulation. The parameters will be assigned from the ff99SB force field.

11. To save the parm7 and rst7 file use the saveamberparm command.

Warning:
Pay close attention to the output of this command for any warnings or errors that your parm7 and rst7 file did not build correctly.

Amber parameter/topology and coordinate files

The alanine dipeptide parm7 and input files are available here:
parm7
rst7

Quit xLEaP

12. To quit xLEaP use quit. The last components needed are the input files that define the program settings for each MD run. For this system, we will perform an energy minimization on the system, then slowly heat the system, and then do production MD at the desired temperature and pressure.

1. Minimization
2. Heating with constant volume and temperature (NVT) for 20ps from 0K to 300K
3. Production MD with constant pressure and temperature (NPT) at 300K and 1atm for 60ps

We will save the trajectory and write to the output file every 2ps. The Langevin thermostat will be used to control the temperature. The random number generator will be initialized with a random seed.
To control all these settings, we will write a simple input file in a text editor. Linux has many text editors available, but we will use a simple text editor included on your linux computer.
13. Open the gedit Text Editor on your Linux computer.
gedit's interface looks like this:

Minimization input

14. Create the file 01_Min.in that includes the following settings for minimization: The settings can be summarized as follows:

imin=1	Choose a minimization run
ntx=1	Read coordinates but not velocities from ASCII formatted rst7 coordinate file
irest=0	Do not restart simulation. (not applicable to minimization)
maxcyc=2000	Maximum minimization cycles
ncyc=1000	The steepest descent algorithm for the first 0-ncyc cycles, then switches the conjugate gradient algorithm for ncyc-maxcyc cycles
ntpr=100	Print to the Amber mdout output file every ntpr cycles
ntwx=0	No Amber mdcrd trajectory file written (not applicable to minimization)
cut=8.0	Nonbonded cutoff distance in Angstroms (for PME, limit of the direct space sum - do NOT reduce this below 8.0. Higher numbers give slightly better accuracy but at vastly increased computational cost.)

Heating input

15. Create the file 02_Heat.in that includes the following settings for heating: The settings can be summarized as follows:

imin=0	Choose a molecular dynamics (MD) run [no minimization]
nstlim=10000	Number of MD steps in run (nstlim * dt = run length in ps)
dt=0.002	Time step in picoseconds (ps). The time length of each MD step
ntf=2	Setting to not calculate force for SHAKE constrained bonds
ntc=2	Enable SHAKE to constrain all bonds involving hydrogen
tempi=0.0	Initial thermostat temperature in K (see NMROPT section)
temp0=300.0	Final thermostat temperature in K (see NMROPT section)
ntwx=1000	Write Amber trajectory file mdcrd every ntwx steps
ntb=1	Periodic boundaries for constant volume
ntp=0	No pressure control
ntt=3	Temperature control with Langevin thermostat
gamma_ln=2.0	Langevin thermostat collision frequency
nmropt=1	NMR restraints and weight changes read (see NMROPT section)
ig=-1	Randomize the seed for the pseudo-random number generator [always a good idea unless you are debugging a simulation problem]

Thermostat temperature via NMROPT

The final three lines allow the thermostat to change its target temperature throughout the simulation. For the first 9000 steps, the temperature will increase from 0K to 300K. For steps 9001 to 10000, the temperature will remain at 300K.

Production input

Warning:
By itself, this input file is not intended for general MD simulations.
NTPR and NTWX are set very low so that it is possible to analyze this short simulation. Using these settings for longer MD simulations will create very large output and trajectory files and will be slower than regular MD. For real production MD, you'll need to increase NTPR and NTWX.
The production time of this simulation is only 60ps. Ideally, we would run this simulation for much longer, but in the interest of time for this tutorial, we have limited the production simulation time.

16. Create the file 03_Prod.in with the settings for production MD:The settings for production can be summarized as follows:

ntx=5	Read coordinates and velocities from unformatted rst7 coordinate file
irest=1	Restart previous MD run [This means velocities are expected in the rst7 file and will be used to provide initial atom velocities]
temp0=300.0	Thermostat temperature. Run at 300K
ntb=2	Use periodic boundary conditions with constant pressure
ntp=1	Use the Berendsen barostat for constant pressure simulation

Input files

The sander input files are available here:
01_Min.in
02_Heat.in
03_Prod.in
Now that we have all the ingredients: the parameter and topology file parm7, the coordinate file rst7

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, and the input files 01_Min.in, 02_Heat.in, 03_Prod.in, we are ready to run the actual minimization, heating, and production MD.
To do this, we will use the program sander, the general purpose MD engine of Amber (there is also a high performance version, termed pmemd, which is part of the commercial version of AMBER and the optimum choice of MD engine but for purposes of the tutorial sander will suffice). sander is run from the command line. On the command line, we can specify several more options and choose which files are to be used for input.
17. First, from the terminal, you will need to change directories to the Tutorial directory with all of your input files. '~' is a shortcut to your home directory where you created a Tutorial directory.

Run minimization

18. Run the minimization of alanine dipeptide with sander. sander uses a consistent syntax for each step of MD simulation. Here is a summary of the command line options of sander:

-O	Overwrite the output files if they already exist
-i 01_Min.in	Choose input file (default mdin)
-o 01_Min.out	Write output file (default mdout)
-p parm7	Choose parameter and topology file parm7
-c rst7	Choose coordinate file rst7
-r 01_Min.ncrst	Write output restart file with coordinates and velocities (default restrt)
-inf 01_Min.mdinfo	Write MD info file with simulation status (default mdinfo)

sander should complete the minimization in a moderate amount of time (~ 27 seconds) depending on your computer specifications.
After sander completes, there should be an output file 01_Min.out, a restart file 01_Min.ncrst, and a MD info file 01_Min.mdinfo. You will use the restart file 01_Min.ncrst for the heating of the system.

Minimization output files

The minimization output files are available here:
01_Min.out
01_Min.ncrst

19. Using gedit, open the outputr file 01_Min.out.
In the 01_Min.out file, you will find the details of your minimization. You should be able to see the system energy ENERGY decrease throughout the minimization.

Run heating MD

Now, using the restart file from the initial minimization, we will heat the system.

20. Run the heating of alanine dipeptide with sander.

Here is a summary of the command line options for sander:

-c 01_Min.ncrst	Now for the input coordinates we choose the restart file from minimization
-x 02_Heat.nc	Output trajectory file for MD simulation (default nc)

sander should complete the heating in a moderate amount of time (~ 2.5 mins) depending on your computer specifications.

Heating output files

The heating output files are available here. Some files are compressed and need to be unzipped.
02_Heat.out
02_Heat.ncrst
02_Heat.nc
21. Open the output file 02_Heat.out to view the system output.
In the 02_Heat.out file you will find the output from the heating MD. You should be able to see system information including timestep energies, and temperature. For example on the 1000 time step:
Some of the important values include:

NSTEP	The time step that the MD simulation is at
TIME	The total time of the simulation (including restarts)
TEMP	System temperature
PRESS	System pressure
Etot	Total energy of the system
EKtot	Total kinetic energy of the system
EPtot	Total potential energy of the system

Note that the pressure is 0.0 because the barostat (pressure control) is not being used in the heating.

Run production MD

Now that minimization and heating are complete. We move on to the actual production MD.
22. Run the production MD of alanine dipeptide with sander.Note: With the '&' at the end of the command, sander now runs in the background
Now sander is running in the background. It will take some time to run the production MD.
But we'd like to monitor the status of the production MD. So we will monitor the sander output file to check the status of the run. The Linux program tail will print the end of the output file.

23. To monitor the status of this job use the program tail to print the output file to the terminal. This prints the output file as sander is running. It's useful to keep track of the job. You can also monitor the mdinfo file (cat 03_Prod.info) which provides detailed performance data as well as estimated time to completion.
24. To exit tail, quit the program by pressing <CTRL-C>.

Complete the MD simulation

Let the MD simulation run. It should take a while to complete the simulation [~ 10 minutes].

Production output

The production MD output is available here:
03_Prod.out
03_Prod.ncrst
03_Prod.nc
Once it's complete open the output file to check that the simulation completed normally.
25. Open the output file 03_Prod.out with gedit to view the output of the MD simulation.
You've now run an MD simulation. In order to visualize the results, we will now use a program called VMD (Visual Molecular Dynamics). This is a molecular graphics program that can render molecular structures in 3D. VMD not only loads Protein Database (PDB) structure files, but also MD trajectories from many programs. [A more in-depth tutorial on VMD is available as an optional hands-on session listed at the bottom of the workshop program page].
26. To start VMD, open a terminal, change directory to your tutorial files in the Tutorial directory, and run vmd. Remember ~/Tutorial is a shortcut to your Tutorial directory. VMD should look like this:
VMD is a very useful tool to visualize protein, nucleic acid, and other biomolecular atomic structures. One of the most common formats is the PDB biomolecular structure format. To load a PDB, got to File -> New Molecule... Then load files for a New Molecule and choose the PDB file. VMD should determine the file type Automatically.
However, we would like to visualize the alanine dipeptide trajectory. Now we will load our MD trajectory to look at the dynamics of alanine dipeptide.
27. Create a new molecule with File -> New Molecule..
28. Load files for New Molecule. Then choose the Amber parameter and topology file parm7. Set the file type to AMBER7 Parm. Click Load.
29. Load files for 0: parm7. Then choose the Amber trajectory file 03_Prod.nc. Set the file type to NetCDF (AMBER, MMTK). Click Load.
VMD now loads your trajectory to be visualized. The main VMD window can be used to control playback.
You should be able to see the alanine dipeptide molecule as well as the many water molecules in the display. You can rotate, zoom and pan the molecules around the display with the mouse.
Many different visualization options can be changed in the Graphics -> Representations window.
Your visualization can be restricted to the alanine dipeptide as well.
30. Change the Selected Atoms to all not water.
You can change the drawing method for the molecule to a more interesting model.
31. Change the Drawing Method to Licorice.
Alanine dipeptide will look something like this:

More VMD

VMD has a lot of functions that can be used to analyze and study a MD trajectory. For example it is possible to align molecules, measure root mean squared deviations (RMSD), save structures from a trajectory, and measure physical system parameters throughout a trajectory. It's also possible to render a movie of a trajectory.
However, these functions are beyond the scope of this introductory section. For more details refer to the VMD tutorial on the main AMBER Tutorial page. Amber includes a suite of tools to examine and analyze MD trajectories. In this tutorial, we will do a simple analysis with several Amber programs and plot the results. The analysis will primarily be done from the command line in the terminal.
32. Open a terminal and change directory to your tutorial files.33. Make an Analysis directory and change to that directory. Now we will use an analysis script process_mdout.perl to analyze the MD output files. This script will extract the energies, temperature, pressure, density, and volume from the MD output files and save them to individual data files.
34. Process the MD output files with process_mdout.perl It is now quite simple to plot the data saved in the output files.
We will use a convenient, simple plotting program called xmgrace to automatically generate plots for the following MD simulation properties throughout the simulation. We use this for our convenvience, but you can use any plotting program of your choice.

1. MD simulation temperature
2. MD simulation density
3. MD simulation total, potential, and kinetic energies.

However, for the MD simulation density, the heating portion of the simulation does not include a density output. You will need to edit the summary.DENSITY file to remove the empty data points for xmgrace to work.
35. Use gedit to edit the summary.DENSITY file to remove the empty data points (up to 20ps).
36. Plot these properties through the simulation using the following commands.

Analysis data files

The analysis data files are available here:
summary.TEMP
summary.DENSITY
summary.ETOT
summary.EPTOT
summary.EKTOT

Production MD system properties

Warning:
We should run this simulation much longer so that the density has equilibrated and the simulation converges. However, in the interest of time for this tutorial, the production MD simulation time has been set very low so that it possible to analyze the results.
The resulting plots should look similar to these:
Alanine dipeptide MD temperature Here you can see the linear increase in temperature during heating (0-20ns). This is followed by the relatively stable temperature fluctuations about 300 K during the production simulation.
Alanine dipeptide MD density During the 20-80ps, the density equilibrates to approximately 1 g/cm^3. This corresponds to a change in periodic box dimensions as the density of the system converges.
Alanine dipeptide MD total, potential, and kinetic energy This plot shows the total system energy which can be decomposed to the total potential energy and the total kinetic energy.
The root mean squared deviation (RMSD) value is a measurement of how similar a structure's internal atomic coordinates are relative to some reference molecule coordinates. For this example, we will measure how the internal atomic coordinates change relative to the minimized structure. Specifically, we will analyze the alanine atoms (residue 2).
To do this analysis, we will use cpptraj, a fairly comprehensive analysis program for processing MD trajectories. This program runs simple user-written scripts that choose what trajectories to load, what analysis to run, and what processed trajectories or structure to save.
First, we will need to write a simple cpptraj script to do this analysis.
37. Use gedit to create a cpptraj script called rmsd.cpptraj. This is a brief summary of the cpptraj script:

trajin 02_Heat.nc	Load the trajectory 02_Heat.nc
reference 01_Min.ncrst	Define the structure 01_Min.ncrst as the reference structure
center :1-3 mass origin	Center the residues 1-3 by mass to the system origin
image origin center	Image the atoms to the origin using the center of mass of the molecule
rms reference mass out 02_03.rms time 2.0 :2	Calculate the mass weighted RMSD using the reference and output to 02_03.rms

Cpptraj input script file

The cpptraj input script file is available here:

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rmsd.cpptraj

Run cpptraj

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To actually run cpptraj, we must run it again from the terminal in the directory where the parm7, nc, and reference ncrst file is located.
38. Using the terminal, change directory to your Tutorial folder, and run cpptraj. A parm7 file and cpptraj script must be specified. Now our RMSD data is stored in the file 02_03.rms. We can simply plot this file with

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xmgrace.
39. Plot the RMSD with xmgrace.

Cpptraj output files

The cpptraj output files are available here:
02_03.rms
cpptraj.log
Alanine dipeptide MD RMSD relative to minimized initial structureIn this example, there is no significant conformational change in the n the Phi/Psi dihedral angles in the alanine dipeptide. This indicates a more stable peptide structure. Congratulations. You've now run your first complete MD simulation and successfully analyzed the results. This is a fairly simple example of the workflow for setting up, running, and analyzing your own MD simulation. If you want to learn more you are encouraged to complete the additional tutorials on the AMBER website. All of the files for the tutorial are available here:
Alanine_Dipeptide_Files.zip