Methods of Molecular Dynamics and Monte Carlo

Introduction to Gromacs 2016

Hector Martinez-Seara, Lukasz Cwiklik

Intitute of Organic Chemistry and Biochemistry

Czech Academy of Sciences

29/11/2017

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1 Molecular mechanics (MD)
Basics
Force field
Workflow
2 What is gromacs and when to use it?
Description
Technical characteristics
3 How to use gromacs?
Gromacs general usage
Instructions Tutorial
Task01: Water box simulation
Task02: GFP protein in water simulation
Task03: Membrane simulation using CHARMMGUI
Task04: Simulation of a transmembrane protein using Martini
4 Appendixes
Commands cheatsheet
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 Molecular mechanics (MD) Basics

How ”classical“ molecular dynamics work?

Degrees of freedom:
atoms/beads are the
elementary particles

Ideal for biological systems

Forces or
interactions Boundary where important interactions do
between particles conditions
not involve reforming of bonds.
Molecular The time evolution of the
Model
system could be obtained by
Force field =
physicochemical
system
temperature
numerically integrating the
knowledge pressure
walls
Newton’s equations of motion.
external forces
The heart of the simulation is
the force field.
Methods to generate
configurations of
atoms: Newton

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 Molecular mechanics (MD) Force field

What is Molecular Dynamics?

Molecular Mechanics (MD) is a methodology that allows to follow the

position of any given number of atoms or particles (beads) (1 ... N)
with the time assuming that their trajectories can be described
accurately in the classical context (Newton’s Laws).

d 2 ri
mi = Fi = −∇ri U(r1 , r2 , . . . , rN ) (1)
dt 2

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 Molecular mechanics (MD) Force field

What is a force Field?

Force fields are normally assumed to consist of pairwise-additive

potentials
More accurate descriptions may involve the use of three-body terms or
non-additive potentials.
A compromise between their accuracy and simplicity is essential.
Most force fields can be described as:

U = Ubond + Uang + Utors + Uvdw + Ucoul (2)

Bonded interactions: bond stretching, angle bending and torsional

dihedral energy contributions for atoms separated by 1, 2 and 3 bonds.
Non Bonded interactions: Van der Waals and Coulomb energy
contributions. These forces apply to all atom pairs.

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 Molecular mechanics (MD) Force field

Typical potentials used in biosystems

Bond Improper

Angle Coulomb

Dihedral Van der Waals

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 Molecular mechanics (MD) Workflow

Typical Molecular dynamics workflow

Define unit cell

Assemble the system
Add solvent
Add ions
Force Field Topology Minimize MD-parameters

Molecule Definition System Definition Simulation Trajectory

Coordinates Analysis

Homology model X-ray, NMR strcture

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 What is gromacs and when to use it? Description

What is GROMACS?

In short
GROMACS is a versatile package to perform molecular dynamics, i.e.
simulate the Newtonian equations of motion for systems with hundreds to
millions of particles.

Area of usage
It is primarily designed for biochemical molecules like proteins, lipids and
nucleic acids that have a lot of complicated bonded interactions, but since
GROMACS is extremely fast at calculating the nonbonded interactions
(that usually dominate simulations) many groups are also using it for
research on non-biological systems, e.g. polymers.

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 What is gromacs and when to use it? Technical characteristics

Major characteristics of GROMACS - I

Extremely high performance compared to all other MD programs.

Excellent CUDA-based GPU acceleration on GPUs.
User-friendly, with topologies and parameter files written in clear text
format.
There is no scripting language - just simple command line options.
Well documented.
Both input files and trajectories are independent of hardware used to
generate them.

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 What is gromacs and when to use it? Technical characteristics

Major characteristics of GROMACS - II

Provides a very compact way of storing trajectory data.

Offers a large selection of flexible tools for trajectory analysis - you
won’t have to write any code to perform routine analyses.
Output trajectories are supported by most of the typical third parties
codes which are used to visualize and analyze the code.
High parallelization capabilities, e.g. MPI, OPENMP, hybrid mode.
The package includes a fully automated topology builder for proteins,
even multimeric structures.
GROMACS is Free Software, available under the GNU Lesser General
Public License.

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 What is gromacs and when to use it? Technical characteristics

Major force fields for MD simulations of biological systems

in gromacs

All Atom
ff Protein Membrane Sugars DNA/RNA
charmm charmm36 charmm36 charmm36 charmm36
amber amber99sb-ildn slipids glycam06h amber99sb-ildn
oplsaa oplsaa Macrog (very basic) no

United Atom
ff Protein Membrane Sugars DNA/RNA
gromos yes yes (very basic) yes

Coarse Grained
ff Protein Membrane Sugars DNA/RNA
martini yes yes (very basic) yes

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 How to use gromacs? Gromacs general usage

The gmx prefix

gmx prefix
Starting from gromacs 5 you should invoke all the tools using the gmx
prefix. Starting from version 5.1 this is in fact the only way to access
gromacs tools

Executing gmx
gmx [-[no]h] [-[no]quiet] [-[no]version] [-[no]copyright] [-nice <int>]
[-[no]backup]

The most useful gmx command

gmx help

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 How to use gromacs? Gromacs general usage

Listing the available gromacs tools

The most useful gmx command

gmx help commands

LIST OF AVAILABLE COMMANDS

Usage: gmx [<options>] <command> [<args>]

Available commands:
anadock Cluster structures from Autodock runs
anaeig Analyze eigenvectors/normal modes
analyze Analyze data sets
angle Calculate distributions and correlations for angles and dihedrals
bar Calculate free energy difference estimates through Bennett’s acceptance ratio
bundle Analyze bundles of axes, e.g., helices
check Check and compare files
... ...

Gromacs v5.1.4 has around 100 tools already at your service!!

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 How to use gromacs? Gromacs general usage

When you should use gromacs tools?

A free advise
Do not reinvent the wheel. Check before starting creating your own tools.
The chances that gromacs has what you need is really high. And likely it
will do it better and faster that you can!! This will save a lot of your
valuable time.

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 How to use gromacs? Gromacs general usage

Running a gromacs tools

The most useful gmx command

gmx help <command>

gmx help solvate

SYNOPSIS
gmx solvate [-cp [<.gro.g96...>]] [-cs [<.gro.g96...>]] [-p [<.top>]] [-o [<.gro.g96...>]] [-box <vector>] [-radius <real>]
[-scale <real>] [-shell <real>] [-maxsol <int>] [-[no]vel]

DESCRIPTION
gmx solvate can do one of 2 things:
1) Generate a box of solvent. Specify -cs and -box. Or specify -cs and -cp with a structure file with a box, but without atoms.
2) Solvate a solute configuration, e.g. a protein, in a bath of solvent
...

OPTIONS
Options to specify input files:
-cp [<.gro/.g96/...>] (protein.gro) (Opt.) Structure file: gro g96 pdb brk ent esp tpr
-cs [<.gro/.g96/...>] (spc216.gro) (Lib.) Structure file: gro g96 pdb brk ent esp tpr
Options to specify input/output files:

...

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 How to use gromacs? Instructions Tutorial

Tasks: General Instructions

General Instructions
Only use the folder provided to perform the tasks included in this
course.
cd $HOME/task-material/
Do not change the file names used in the tasks.
In real life this is not necessary but it is to minimize wasting time
during this course.
All exercises provide solution files prefixed with ”EX”.
Never modify these files. They are provided as reference only.
Before using them copy them:
cp EX-$FILENAME $FILENAME
If you are stuck ask for help immediately to the professors.

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 How to use gromacs? Task01: Water box simulation

Task01: Simulation of a water box with/without ions

Purpose of the task

To introduce the general workflow of a Molecular dynamics
simulation. Many concepts introduced here are shared among MD
engines. Only the commands used differ.
To show all the stages required to produce scientific quality data
when using MD simulations
1 Minimization - To remove bad contacts produced in the assembling of
the system
2 Equilibration - Simulation to allow the system to the correct
equilibrium simulation ensemble.
3 Production - The stage where we produce the real data from our
system.

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 How to use gromacs? Task01: Water box simulation

Generation and visualization of a water box

Water box generation:

Using gmx solvate generate a TIP3P
water box of 5x10x7 namometers
1 cd task01-water
2 gmx solvate -cs -box 5 10 7 -o
00-water.gro
(Copy the number of waters for later use!!)

Inspect the content of the gro file in:

e.g. vim, nano, emacs,...
Visualize the gro file: vmd water.gro
1 Change the representation to
orthographic
2 Draw the simulation box:
Execute pbc box in the
console execute
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 How to use gromacs? Task01: Water box simulation

Water box simulation - Minimization

Minimization protocol:
Water box
1 cp EX-01-min.mdp 01-min.mdp -3
Minimization

Potential
2 cp EX-00-water.top 00-water.top -3.5

(change number of waters if required) -4

(kJ/mol)* 1E5
-4.5
3 gmx grompp -c 00-water.gro
-5
-f 01-min.mdp -p 00-water.top
-5.5
-o 01-water-min.tpr
-6
0 50 100 150 200
-po 01-mdout-min.mdp Time (ps)

4 gmx mdrun -v -deffnm 01-water-min

Important step to avoid possible initial
5 echo ”Potential” |gmx energy
bad contacts
-f 01-water-min.edr -o 01-energy-min.xvg
6 xmgrace -free -nxy 01-energy-min.xvg

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 How to use gromacs? Task01: Water box simulation

Water box simulation - MD Equilibration

Equilibration protocol:
1 cp EX-01-water-min.gro 01-water-min.gro
Water box
Equilibration
2 cp EX-02-eq.mdp 02-eq.mdp (notice that 400

we use berendsen)
300
3 gmx grompp -c 01-water-min.gro
-f 02-eq.mdp -p 00-water.top

T (K)
200
-o 02-water-eq.tpr -po 02-mdout-eq.mdp
4 gmx mdrun -v -deffnm 02-water-eq 100
Temperature
5 echo ”Temperature” |gmx energy
-f 02-water-eq.edr 0
0 20 40 60 80 100
Time (ps)
-o 02-temperature-eq.xvg
6 xmgrace -free -nxy 02-temperature-eq.xvg

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 How to use gromacs? Task01: Water box simulation

Water box simulation - MD Simulation

Production simulation protocol: Water box

Production
1 cp EX-02-water-eq.gro 02-water-eq.gro
2 cp EX-03-md.mdp 03-md.mdp Density
975

3 gmx grompp -c 02-water-eq.gro

(kg/m^3)
-f 03-md.mdp -p 00-water.top 970
-o 03-water-md.tpr -po 03-mdout-md.mdp
4 gmx mdrun -v -deffnm 03-water-md 965

5 echo ’Density’ | gmx energy

-f 03-water-md.edr -o 03-density-md.xvg 960
0 200 400 600 800 1000
Time (ps)
6 xmgrace -free -nxy 03-density-md.xvg

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 How to use gromacs? Task01: Water box simulation

Water box simulation - Visualization MD Simulation

Visualization protocol:
1 Fix broken molecules across periodic boundary
conditions
gmx trjconv -f 03-water-md.xtc -s 03-water-md.tpr
-o 03-water-md-mol.xtc -pbc mol
2 Visualize water box in VMD
vmd 02-water-eq.gro 03-water-md-mol.xtc
Visualize the hydrogen bond formation destruction
during the simulation.
NOTE: Select ”Update Selection Every Frame”

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 How to use gromacs? Task01: Water box simulation

Water box with ions - Add ions to water

Salt addition protocol:

1 cp EX-03-water-md.gro 03-water-md.gro
2 cp EX-00-water.top 04-ions.top
3 gmx grompp -c 03-water-md.gro -f 03-md.mdp
-p 00-water.top -o 04-water-genion.tpr
-po 04-mdout-genion.mdp
4 gmx genion -s 04-water-genion.tpr -p 04-ions.top
-o 04-NACL-solution-genion.gro -conc 0.150 (Select ”SOL”)

5 vmd 04-NACL-solution-genion.gro
Create separate representations for water (lines)
and ions (VDW)

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 How to use gromacs? Task01: Water box simulation

Water box with ions - Simulation

Simulation protocol (use the mdp files from the water simulation):
1 Minimization:
1 gmx grompp -f 01-min.mdp -c 04-NACL-solution-genion.gro -p 04-ions.top
-o 05-ions-min.tpr -po 05-ions-min-mdout.mdp
2 gmx mdrun -v -deffnm 05-ions-min
2 Equilibration:
1 gmx grompp -f 02-eq.mdp -c 05-ions-min.gro -p 04-ions.top -o 06-ions-eq.tpr
-po 06-ions-eq-mdout.mdp
2 gmx mdrun -v -deffnm 06-ions-eq
3 Production simulation:
1 gmx grompp -f 03-md.mdp -c 06-ions-eq.gro -p 04-ions.top -o 07-ions-md.tpr
-po 07-ions-md-mdout.mdp
2 gmx mdrun -v -deffnm 07-ions-md

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 How to use gromacs? Task01: Water box simulation

Water box with ions - Visualization

Visualization protocol of 07-ions-md.xtc trajectory:

1 Fix broken molecules across periodic boundary conditions:
gmx trjconv -f 07-ions-md.xtc -s 07-ions-md.tpr -o 07-ions-md-mol.xtc
-pbc mol
2 Visulaize trajectory with VMD
vmd 06-ions-eq.gro 07-ions-md-mol.xtc
Create separate representations for water (lines) and ions (VDW)
Visualize the hydrogen bond formation destruction during the
simulation
Analyze visually the first hydration shell of a selected cation and anion:
same resname as (water within 5 of resid ”ION-ID”)
NOTE: Select ”Update Selection Every Frame”

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 How to use gromacs? Task01: Water box simulation

Analyzing simulation

Time dependencies: energy, temperature, pressure

1 gmx energy -f 07-ions-md.edr -o PROPERTY.xvg
2 xmgrace -free -nxy PROPERTY.xvg

Radial distribution function of water around Na+ and Cl−

1 gmx rdf -f 07-ions-md.xtc -s 07-ions-md.tpr -o 07-ions-md-ana-rdf-NA.xvg -ref NA -sel SOL
2 gmx rdf -f 07-ions-md.xtc -s 07-ions-md.tpr -o 07-ions-md-ana-rdf-CL.xvg -ref CL -sel SOL
3 xmgrace 07-ions-md-ana-rdf-NA.xvg 07-ions-md-ana-rdf-CL.xvg -free -legend load

Water diffusion coefficient - Mean Squared displacement (MSD):

1 gmx msd -f EX-07-ions-md.xtc -s EX-07-ions-md.tpr -o EX-07-ions-md-ana-msd-water.xvg
-rmcomm (How does compare with the experimental value: 2.9x10−5 cm2 /s)

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 How to use gromacs? Task02: GFP protein in water simulation

Task02: Simulation Green Fluorecence protein (GFP)

Purpose of the task

In this task we will simulate the GFP protein in solution (PDB: 1GFL).
Molecular dynamics is an excellent tool to study protein behavior. For
these reasons many MD works actually focus on proteins.
Most of the concepts introduced here can be applied to the simulation
of other biomolecules with only small adaptations/modifications.

Change to the new task directory

cd ../task02-GFP

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 How to use gromacs? Task02: GFP protein in water simulation

Where to start?

Define unit cell

Assemble the system
Add solvent
Add ions
Force Field Topology Minimize MD-parameters

Molecule Definition System Definition Simulation Trajectory

Analysis
Coordinates

Homology model X-ray, NMR strcture

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 How to use gromacs? Task02: GFP protein in water simulation

Obtaining the protein structure

When simulating biomolecules establishing a

reasonable position of its constituent atoms
is not a trivial task:
They usually contain hundreds or even
million of atoms.
Their connectivity, stereochemistry and
spacial distribution is very complicated.

Obtaining protein structure

RCSB Protein Data Bank
http://www.rcsb.org/

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 How to use gromacs? Task02: GFP protein in water simulation

Download the pdb of the GFP protein

Obtaining GFP protein - 1GFL

Download 1GFL from http://www.rcsb.org/
(save it as 01-GFP-topology/00-1GFL.pdb)
Open 00-1GFL.pdb in your favorite text editor
Visualize it in vmd
Display secondary structure information
by changing color and representation
Display different amino acids types

Observations:
1 There are two different chains
2 There are few crystallized waters
3 Read REMARKS 350 465 470!!
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 How to use gromacs? Task02: GFP protein in water simulation

Selecting a force field

Define unit cell

Assemble the system
Add solvent
Add ions
Force Field Topology Minimize MD-parameters

Molecule Definition System Definition Simulation Trajectory

Analysis
Coordinates

Homology model X-ray, NMR strcture

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 How to use gromacs? Task02: GFP protein in water simulation

Selection of a decent protein force field

All atom
charmm36, amber99sb-ildn , oplsaa, charmm27

United atom
GROMOS96 54a7

Coarse-grained
Martini
The force field to be selected for this task
Not good but seldom used.
Never good – You MUST know what are you doing.

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 How to use gromacs? Task02: GFP protein in water simulation

Task02: Generating the protein topology

Define unit cell

Assemble the system
Add solvent
Add ions
Force Field Topology Minimize MD-parameters

Molecule Definition System Definition Simulation Trajectory

Coordinates Analysis

Homology model X-ray, NMR strcture

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 How to use gromacs? Task02: GFP protein in water simulation

gmx pdb2gmx - your best/worst friend

gmx pdb2gmx
Tool provided by gromacs to convert coordinate files to topology and force
field compliant coordinate files. Mainly used for proteins.

gmx pdb2gmx reads a .pdb (or .gro) file, reads some database files, adds
hydrogens to the molecules and generates coordinates in GROMACS
format and a topology in GROMACS format. These files can subsequently
be processed to generate a run input file.

Normal execution
gmx pdb2gmx -f protein.pdb

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 How to use gromacs? Task02: GFP protein in water simulation

GFP topology - Raw attempt

Try to generate the topology for 00-1GFL.pdb using pdb2gmx using amber99sb-ildn
using tip3p water model

Forced execution Normal execution

gmx pdb2gmx -f 00-1GFL.pdb gmx pdb2gmx -f 00-1GFL.pdb
-missing -o 00-1GFL -p 00-1GFL
WARNING: atom O is missing in residue
-i 00-1GFL
SER 65 in the pdb file.
WARNING: atom O is missing in residue SER Fatal error: There were 1 missing atoms in molecule

65 in the pdb file. Protein-chain-A, if you want to use this incomplete

WARNING: Residue 65 named SER of a molecule in the input topology anyhow, use the option -missing

file was mapped to an entry in the topology database, but the OPTION:
atom O used in an interaction of type improper in that entry is -[no]missing (no) Continue when atoms are missing,

not found in the input file. Perhaps your atom and/or residue dangerous

naming needs to be fixed.

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 How to use gromacs? Task02: GFP protein in water simulation

GFP topology - Preprocess original pdb

Clean 00-1GFL.pdb
1 Remove all HETATM lines
grep -v ”HETATM” 00-1GFL.pdb > 01-1GFL-noHETATM.pdb
Be aware that some nonstandards residues might be input as
HETATM, e.g. Selenomethionine.
2 Remove chain B from pdb (for convenience of the exercise)
grep -v ” B ” 01-1GFL-noHETATM.pdb >02-1GFL-A-raw.pdb
Notice that B is surrounded by one space in each side

This is a very crude method. It is often better to open the pdb with a
raw text editor (e.g. vi, emacs, nano) and do these tasks manually.

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 How to use gromacs? Task02: GFP protein in water simulation

GFP topology - Clean pdb (”Extra”)

pdb4amber
Tool shiped with ambertools which prepare pdbs for further
processing in tleap which add missing heavy atoms.
Among its capabilities it can remove waters, create Disulfide bonds,
estimate the protonation state bases on the hydrogen atoms in the
histidines . . .

Prepare pdb for ambertools using pdb4amber

pdb4amber -i 02-1GFL-A-raw.pdb -o 03-1GFL-pdb4amber.pdb --reduce --dry

Notice it says: ”Missing Heavy Atoms (Renumbered Residues!) None”

Try to make pdb2gmx using -ignh

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 How to use gromacs? Task02: GFP protein in water simulation

GFP topology - Add missing heavy atoms (”Extra”)

Correct missing atoms with tleap (part of ambertools suite)

1 tleap
2 source leaprc.ff14SB
3 x = loadPdb 03-1GFL-pdb4amber.pdb
4 savepdb x 04-1GFL.pdb
5 quit

This method will not correct for missing residues. For this you can
use Modeller which can also be used to correct for missing atoms.

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 How to use gromacs? Task02: GFP protein in water simulation

GFP topology - Generate working topology

Generate topology GFP

Use gmx pdb2gmx to generate the topology for 04-1GFL.pdb using tip3p
water model and amber99sb-ildn force field

Normal execution - Problems with hydrogens (FAILS!!)

gmx pdb2gmx -f 04-1GFL.pdb -o 04-1GFL.gro -i 04-1GFL.itp -p 04-1GFL.top
(Hydrogen order is force field dependent)

Normal execution but ignoring pdb hydrogens (WORKS!!)

gmx pdb2gmx -f 04-1GFL.pdb -o 04-1GFL.gro -i 04-1GFL-posres.itp -p 04-1GFL.top
-n 04-1GFL.ndx -ignh
Notice that the total charge of the protein is -5 e

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 How to use gromacs? Task02: GFP protein in water simulation

GFP topology - Isolate 1GFL molecule topology

For convenience and clarity we usually isolate molecules topologies from

the system topology. This allows the recycling of the generated topologies
and simplify the system topology file.

Prepare GFP topology file: 1GFL.itp

Extract the 1GFL.itp from 04-1GFL.top using text editor
cp 04-1GFL.top 04-1GFL.itp
Open 1GFL.itp in using your favorite raw text editor (vim, nano, emacs
...)
Remove until ”[ Moleculetype ]” from 1GFL.itp
Change name ”[ Moleculetype ]” from ”Protein” to ”1GFL”
Remove everything from ”; Include water topology”
Save the newly created 1GFL topology file

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 How to use gromacs? Task02: GFP protein in water simulation

The molecule topology file - How itp files look like?

The itp files - Topologies of Molecules

[ moleculetype ] ← Tag that marks the beginning of a molecule definition
; Name nrexcl ← This is a comment which starts ”;”
Protein 3 ← Do not compute long range interaction for atoms 3 bonds apart
[ atoms ]
; nr type resnr residue atom cgnr charge mass typeB chargeB massB
1 N3 1 ALA N 1 0.1414 14.01 ; qtot 0.1414
...
[ bonds ] ← Funct type depends on the force field used
; ai aj funct c0 c1 c2 c3
1 2 1 ← last number, i.e. 1, refers to a simple harmonic potential. The function used for bond by amber99sb-ildn
...
[ angles ]
; ai aj ak funct c0 c1 c2 c3
2 1 3 1 ← The lack of explicit parameters c0 c1 ... means that default parameters from #include
”amber99sb-ildn.ff/forcefield.itp” file will be used
...
[ dihedrals ]
; ai aj ak al funct c0 c1 c2 c3 c4 c5
21569
...
; Include Position restraint file
#ifdef POSRES ← Topologies can conditionally include other parameters, i.e. define=-DPOSRES in mdp file”
#include ”04-1GFL-posres.itp” ← Restrains file for our protein”
#endif

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 How to use gromacs? Task02: GFP protein in water simulation

GFP topology - The 04-1GFL.itp topology file

[ moleculetype ]
; Name nrexcl
1GFL 3
[ atoms ]
; nr type resnr residue atom cgnr charge mass typeB chargeB massB
; residue 1 ALA rtp NALA q +1.0
1 N3 1 ALA N 1 0.1414 14.01 ; qtot 0.1414
2 H 1 ALA H1 2 0.1997 1.008 ; qtot 0.3411
3 H 1 ALA H2 3 0.1997 1.008 ; qtot 0.5408
...
3576 3593 3591 3592 4
3591 3595 3593 3594 4
3595 3606 3605 3607 4
; Include Position restraint file
#ifdef POSRES
#include ”EX-04-1GFL-posres.itp”
#endif

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 How to use gromacs? Task02: GFP protein in water simulation

Protein definition achieved

− − − − − − − − − − PLEASE NOTE − − − − − − − − − −
We have successfully generated a topology from: 04-1GFL.pdb. The
Amber99sb-ildn force field and the tip3p water model are used.

Define unit cell

Assemble the system
Add solvent
Add ions
Force Field Topology Minimize MD-parameters

Molecule Definition System Definition Simulation Trajectory

Coordinates Analysis

Homology model X-ray, NMR strcture

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 How to use gromacs? Task02: GFP protein in water simulation

How do system topology files look like?

The top files

#include ”amber99sb-ildn.ff/forcefield.itp” ← Force field definition
#include ”Protein.itp” ← Include protein topologies
#include ”amber99sb-ildn.ff/tip3p.itp” ← Include water topology
#include ”amber99sb-ildn.ff/ions.itp” ← Include topology for ions
[ system ]
; Name
GREEN FLUORESCENT PROTEIN
[ molecules ]
; Compound #mols
PROTEIN 1 ← Number of proteins
SOL 1000 ← Number of waters (We do not have any yet!!)

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 How to use gromacs? Task02: GFP protein in water simulation

Prepare system topology file

Prepare system topology file: 05-1GFL-nowater.top

#include ”amber99sb-ildn.ff/forcefield.itp”
#include ”04-1GFL.itp”
#include ”amber99sb-ildn.ff/tip3p.itp”
#include ”amber99sb-ildn.ff/ions.itp”
[ system ]
; Name
GREEN FLUORESCENT PROTEIN
[ molecules ]
; Compound #mols
1GFL 1

NOTE: cp EX-05-1GFL-nowater.top 05-1GFL-nowater.top or use

EX-04-1GFL.top as template.
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 How to use gromacs? Task02: GFP protein in water simulation

GFP in vacuum - Create simulation box

Put GFP protein in a simulation box

1 gmx editconf -f 04-1GFL.gro
-o 05-1GFL-nowater-center.gro -box 6 6 6
2 vmd 05-1GFL-nowater-center.gro

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 How to use gromacs? Task02: GFP protein in water simulation

Generation of coordinates and topologies for protein

simulation in vacuum

Define unit cell

Assemble the system
Add solvent
Add ions
Force Field Topology Minimize MD-parameters

Molecule Definition System Definition Simulation Trajectory

Coordinates Analysis

Homology model X-ray, NMR strcture

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 How to use gromacs? Task02: GFP protein in water simulation

GFP in water - Solvate GFP

Immerse the GFP protein in a box of

water
cp 05-1GFL-nowater.top
06-1GFL-water.top
gmx solvate -cs -p 06-1GFL-water.top
-cp 05-1GFL-nowater-center.gro
-o 06-1GFL-water.gro
vmd 06-1GFL-water.gro

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 How to use gromacs? Task02: GFP protein in water simulation

GFP in water - The parameter file minimization

A typical minimization mdp file: 07-min.mdp

define = -DPOSRES ← Protein restrained
integrator = steep ← We will do a minimization
emtol = 1000.0 ← min energy difference KJ/mol
nsteps = 5000 ← max number steps
nstlist = 10 ← how often update neighbors list
cutoff-scheme = Verlet ← Search neighbor method for nonbonded-interaction
rlist = 1.2 ← force field dependent
vdwtype = Cut-off ← Calculate vdw interactions with atoms inside ”rvdw”
vdw-modifier = Potential-shift-Verlet ← force field dependent
rvdw switch = 1.0 ← force field dependent
rvdw = 1.2 ← force field dependent
coulombtype = pme ← Usage particle mesh Ewald
rcoulomb = 1.2 ← force field dependent
coulomb-modifier = Potential-shift-Verlet ← force field dependent
constraints = none ← No constrains will be used
constraint algorithm = LINCS ← Useless as ”constrains = none”
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 How to use gromacs? Task02: GFP protein in water simulation

GFP in water - Minimization

Removal of bad contacts between water and a constrained GFP

1 gmx grompp -f 07-min.mdp \← Minimization parameters
-p 06-1GFL-water.top \← Topology system
-c 06-1GFL-water.gro \← Coordinates atoms
-o 07-1GFL-water-min.tpr \← Output file for mdrun
-po 07-1GFL-water-min-mdout.mdp ← Full list minimizaton
parameters
2 gmx mdrun -deffnm 07-1GFL-water-min -v
3 vmd 07-1GFL-water-min.gro

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 How to use gromacs? Task02: GFP protein in water simulation

What to do after adding waters and minimize?

Define unit cell

Assemble the system
Add solvent
Add ions
Force Field Topology Minimize MD-parameters

Molecule Definition System Definition Simulation Trajectory

Coordinates Analysis

Homology model X-ray, NMR strcture

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 How to use gromacs? Task02: GFP protein in water simulation

GFP in water - Neutralization of excess charges and

medium ionic force adjustment

What was this note about

gmx grompp -f 07-min.mdp -p 06-1GFL-water.top -c 06-1GFL-water.gro
-o 07-1GFL-water-min.tpr -po 07-1GFL-water-min-mdout.mdp

NOTE 1 [file 06-1GFL-water.top, line 20]:

System has non-zero total charge: -5.000000
It means that GFP protein has more negatively charged amino acids
than positive ones. We need to add cations to neutralize this excess
of charge, e.g. NA+ ions.
Also in biological systems the medium contain salt, e.g. Na+ Cl− ions.

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 How to use gromacs? Task02: GFP protein in water simulation

GFP in water - Adding ions gmx genion

Add salt to the system using gmx solvate

Neutralize and add 150 mMol Na+ Cl− ions.
Do first:
cp 06-1GFL-water.top 08-1GFL-solvated.top
gmx grompp -f 07-min.mdp -c 07-1GFL-water-min.gro -p 06-1GFL-water.top
-o 08-1GFL-genion.tpr -po 08-1GFL-genion-mdout.mdp
Output coordinate file should be called ”08-1GFL-genion.gro”

Solution
gmx genion -s 08-1GFL-genion.tpr -p 08-1GFL-solvated.top -o 08-1GFL-genion.gro
-neutral -conc 0.15
Select ”SOL”
Note: It does not really provide 150 mMol!!

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 How to use gromacs? Task02: GFP protein in water simulation

GFP in water - Minimize GPF system

Minimize system after ions additions

1 gmx grompp -f 07-min.mdp
-p 08-1GFL-solvated.top
-c 08-1GFL-genion.gro
-o 09-1GFL-solvated-min.tpr
-po 09-1GFL-solvated-min-mdout.mdp
2 gmx mdrun -v
-deffnm 09-1GFL-solvated-min
3 vmd 09-1GFL-solvated-min.gro

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 How to use gromacs? Task02: GFP protein in water simulation

GFP in water - Equilibration process

Define unit cell

Assemble the system
Add solvent
Add ions
Force Field Topology Minimize MD-parameters

Molecule Definition System Definition Simulation Trajectory

Coordinates Analysis

Homology model X-ray, NMR strcture

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 How to use gromacs? Task02: GFP protein in water simulation

GFP in water - Equilibration parameters

Equilibration file: 10-eq-pr.mdp Continuation: 10-eq-pr.mdp

define = -DPOSRES ;Protein is restrained tcoupl = berendsen ; nose-hoover
integrator = md tc grps = Protein water and ions
dt = 0.002 tau t = 1.0 1.0
nsteps = 500000 ; 1 ns ref t = 310.00 310.00
nstlog = 500 ; 1ps
nstenergy = 500 ; 1ps pcoupl = berendsen ; Parrinello-Rahman
nstxout-compressed = 500 ; 1ps pcoupltype = isotropic
tau p = 5.0
cutoff-scheme = Verlet compressibility = 4.5e-5
nstlist = 20 ref p = 1.0
rlist = 1.2
constraints = h-bonds
coulombtype = pme
constraint algorithm = LINCS
rcoulomb = 1.2
coulomb-modifier = Potential-shift-Verlet nstcomm = 100
vdwtype = Cut-off comm mode = linear
vdw-modifier = Potential-shift-Verlet refcoord scaling = com
rvdw switch = 1.0
rvdw = 1.2

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 How to use gromacs? Task02: GFP protein in water simulation

GFP in water - Equilibration I

Equilibration restrainded GFP in water

GROMACS Energies
1 gmx grompp -f 10-eq-pr.mdp 215
-c 09-1GFL-solvated-min.gro Volume
214
-p 08-1GFL-solvated.top
-o 10-1GFL-solvated-eq-pr.tpr 213

(nm^3)
-po 10-1GFL-solvated-eq-pr-mdout.mdp 212

2 gmx mdrun -v -deffnm 10-1GFL-solvated-eq-pr 211

3 echo ”Volume” |gmx energy 210

-f 10-1GFL-solvated-eq-pr.edr 209
0 200 400 600 800 1000
Time (ps)
-o 10-1GFL-solvated-eq-pr-Volume.xvg
4 xmgrace 10-1GFL-solvated-eq-pr-Volume.xvg

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 How to use gromacs? Task02: GFP protein in water simulation

GFP in water - Equilibration II (no restrains)

Analysis
Equilibration free GFP in water
1 echo ”Total-Energy” |gmx energy
1 cp 10-eq-pr.mdp 11-eq.mdp
-f 11-1GFL-solvated-eq.edr
2 Comment ”define = -DPOSRES”
-o 11-1GFL-solvated-eq-Total-Energy.xvg
line in 11-eq.mdp with ”;”
2 xmgrace 11-1GFL-solvated-eq-Total-Energy.xvg
3 gmx grompp -f 11-eq.mdp
-2.15
-c 10-1GFL-solvate-eq-pr.gro
-2.155
-p 08-1GFL-solvated.top Total Energy

-2.16
-o 11-1GFL-solvated-eq.tpr
-po 11-1GFL-solvated-eq- (1e+5 kJ/mol) -2.165

mdout.mdp -2.17

4 gmx mdrun -v -2.175

-deffnm 11-1GFL-solvated-eq -2.18

0 200 400 600 800 1000
Time (ps)

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 How to use gromacs? Task02: GFP protein in water simulation

Ready for a the production simulation

Define unit cell

Assemble the system
Add solvent
Add ions
Force Field Topology Minimize MD-parameters

Molecule Definition System Definition Simulation Trajectory

Coordinates Analysis

Homology model X-ray, NMR strcture

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 How to use gromacs? Task02: GFP protein in water simulation

GFP in water - Production parameters

Production file: 12-md.mdp Continuation: 12-md.mdp

define = integrator = md tcoupl = nose-hoover
dt = 0.002 tc grps = Protein water and ions
nsteps = 500000 ; 1ns tau t = 1.0 1.0
nstlog = 5000 ; 10ps ref t = 310.00 310.00
nstenergy = 5000 ; 10ps pcoupl = Parrinello-Rahman
nstxout-compressed = 5000 ; 10ps pcoupltype = isotropic
cutoff-scheme = Verlet tau p = 5.0
nstlist = 20 compressibility = 4.5e-5
rlist = 1.2 ref p = 1.0
coulombtype = pme constraints = h-bonds
rcoulomb = 1.2 constraint algorithm = LINCS
coulomb-modifier = Potential-shift-Verlet
vdwtype = Cut-off nstcomm = 100
vdw-modifier = Potential-shift-Verlet comm mode = linear
rvdw switch = 1.0 refcoord scaling = com
rvdw = 1.2

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 How to use gromacs? Task02: GFP protein in water simulation

System ready for the production simulation

Define unit cell

Assemble the system
Add solvent
Add ions
Force Field Topology Minimize MD-parameters

Molecule Definition System Definition Simulation Trajectory

Coordinates Analysis

Homology model X-ray, NMR strcture

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 How to use gromacs? Task02: GFP protein in water simulation

GFP in water - Production simulation

Production simulation GFP in water

1 cp 11-eq.mdp 12-md.mdp
2 Edit 12-md.mdp:
1 tcoupl = nose-hoover
2 pcoupl = Parrinello-Rahman
3 gmx grompp -f 12-md.mdp -c 11-1GFL-sovated-eq.gro -p 08-1GFL-solvated.top
-o 12-1GFL-solvated-md.tpr -po 12-1GFL-solvated-md-mdout.mdp
4 gmx mdrun -v -deffnm 12-1GFL-solvated-md

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 How to use gromacs? Task02: GFP protein in water simulation

Simulation ready - Are we done?

Define unit cell

Assemble the system
Add solvent
Add ions
Force Field Topology Minimize MD-parameters

Molecule Definition System Definition Simulation Trajectory

Coordinates Analysis

Homology model X-ray, NMR strcture

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 How to use gromacs? Task02: GFP protein in water simulation

GFP in water - Process trajectory

Process trajectory to visualize in vmd

1 gmx check -f 12-1GFL-solvated-md.xtc
2 gmx trjconv -s 12-1GFL-solvated-md.tpr -f 12-1GFL-solvated-md.xtc
-o 12-1GFL-solvated-md-whole.xtc -pbc whole
3 vmd 12-1GFL-solvated-md.gro 12-1GFL-solvated-md-whole.xtc

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 How to use gromacs? Task02: GFP protein in water simulation

Simulation ready, time for analysis

Define unit cell

Assemble the system
Add solvent
Add ions
Force Field Topology Minimize MD-parameters

Molecule Definition System Definition Simulation Trajectory

Coordinates Analysis

Homology model X-ray, NMR strcture

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 How to use gromacs? Task02: GFP protein in water simulation

Analyzing simulation

Radius of gyration:
1 echo ”Protein” | gmx gyrate -f 12-1GFL-solvated-md-whole.xtc
-s 12-1GFL-solvated-md.tpr -o 12-1GFL-solvated-md-ana-gyrate.xvg

Autocorrelation rotational diffusion:

1 printf ”del 0-30 \n a3556|a2224 \n q \n” |gmx make ndx -f 12-1GFL-solvated-md.tpr
-o 12-1GFL-solvated-md-ana-rotacf.ndx
2 gmx rotacf -f 12-1GFL-solvated-md-whole.xtc -s 12-1GFL-solvated-md.tpr
-n 12-1GFL-solvated-md-ana-acf.ndx -o 12-1GFL-solvated-md-ana-rotacf.xvg -d

Radial distribution function of Na+ around GFP

1 gmx rdf -f 12-1GFL-solvated-md-whole.xtc -s 12-1GFL-solvated-md.tpr
-o 12-1GFL-solvated-md-ana-rdf-surf.xvg -surf mol -ref Protein -sel NA

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 How to use gromacs? Task02: GFP protein in water simulation

Gromacs analysis tools I

anadock Cluster structures from Autodock runs

anaeig Analyze eigenvectors/normal modes
analyze Analyze data sets
angle Calculate distributions and correlations for angles and dihedrals
bar Calculate free energy difference estimates through Bennett’s acceptance ratio
bundle Analyze bundles of axes, e.g., helices
chi Calculate everything you want to know about chi and other dihedrals
cluster Cluster structures
clustsize Calculate size distributions of atomic clusters
confrms Fit two structures and calculates the RMSD
covar Calculate and diagonalize the covariance matrix
current Calculate dielectric constants and current autocorrelation function
density Calculate the density of the system
densmap Calculate 2D planar or axial-radial density maps
densorder Calculate surface fluctuations
dielectric Calculate frequency dependent dielectric constants
dipoles Compute the total dipole plus fluctuations
disre Analyze distance restraints
distance Calculate distances between pairs of positions

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 How to use gromacs? Task02: GFP protein in water simulation

Gromacs analysis tools II

do dssp Assign secondary structure and calculate solvent accessible surface area
dos Analyze density of states and properties based on that
dyecoupl Extract dye dynamics from trajectories
dyndom Interpolate and extrapolate structure rotations
enemat Extract an energy matrix from an energy file
freevolume Calculate free volume
gangle Calculate angles
gyrate Calculate the radius of gyration
h2order Compute the orientation of water molecules
hbond Compute and analyze hydrogen bonds
helix Calculate basic properties of alpha helices
helixorient Calculate local pitch/bending/rotation/orientation inside helices
hydorder Compute tetrahedrality parameters around a given atom
mdmat Calculate residue contact maps
mindist Calculate the minimum distance between two groups
mk angndx Generate index files for ’gmx angle’
morph Interpolate linearly between conformations
msd Calculates mean square displacements
nmeig Diagonalize the Hessian for normal mode analysis

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 How to use gromacs? Task02: GFP protein in water simulation

Gromacs analysis tools III

nmens Generate an ensemble of structures from the normal modes

nmtraj Generate a virtual oscillating trajectory from an eigenvector
order Compute the order parameter per atom for carbon tails
pairdist Calculate pairwise distances between groups of positions
polystat Calculate static properties of polymers
potential Calculate the electrostatic potential across the box
principal Calculate principal axes of inertia for a group of atoms
rama Compute Ramachandran plots
rdf Calculate radial distribution functions
rms Calculate RMSDs with a reference structure and RMSD matrices
rmsdist Calculate atom pair distances averaged with power -2, -3 or -6
rmsf Calculate atomic fluctuations
rotacf Calculate the rotational correlation function for molecules
rotmat Plot the rotation matrix for fitting to a reference structure
saltbr Compute salt bridges
sans Compute small angle neutron scattering spectra
sasa Compute solvent accessible surface area
saxs Compute small angle X-ray scattering spectra

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 How to use gromacs? Task02: GFP protein in water simulation

Gromacs analysis tools IV

sham Compute free energies or other histograms from histograms

solvate Solvate a system
sorient Analyze solvent orientation around solutes
spatial Calculate the spatial distribution function
spol Analyze solvent dipole orientation and polarization around solutes
tcaf Calculate viscosities of liquids
traj Plot x, v, f, box, temperature and rotational energy from trajectories
trjorder Order molecules according to their distance to a group
vanhove Compute Van Hove displacement and correlation functions
velacc Calculate velocity autocorrelation functions
Gromacs v5.1.4 has around 100 tools already at your service!!

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 How to use gromacs? Task02: GFP protein in water simulation

GFP simulation complete

Define unit cell

Assemble the system
Add solvent
Add ions
Force Field Topology Minimize MD-parameters

Molecule Definition System Definition Simulation Trajectory

Coordinates Analysis

Homology model X-ray, NMR strcture

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 How to use gromacs? Task03: Membrane simulation using CHARMMGUI

Task03: Membrane simulation using CHARMMGUI

Purpose of the task

Show how tools can simplify the
execution workflow of MD
simulations
Show another type of biological
systems where MD can be
applied

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 How to use gromacs? Task03: Membrane simulation using CHARMMGUI

CHARMMGUI for gromacs

http://www.charmm-gui.org/
CHARMMGUI is a free web resource that will help you to generate input
files for most common biologically relevant systems. It produces files
compatible with Gromacs v5 and uses the modern charmm36 force field

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 How to use gromacs? Task03: Membrane simulation using CHARMMGUI

CHARMMGUI is very efficient to generate biological

membranes
The Input Generator
It provides among other options the possibility to make a personalized
membrane.
Its output can be easily adapted to be used with other force fields not
just charmm36.

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 How to use gromacs? Task03: Membrane simulation using CHARMMGUI

Simulation of a model plasma membrane

Produce a plasma membrane with the following parameters

upper leaflet 60 POPC/60 CHOL /60 PSM
lower leaflet 65 POPE/60 CHOL /60 POPS
default options

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 How to use gromacs? Task03: Membrane simulation using CHARMMGUI

Run a membrane generated by CHARMMGUI

Running CHAMMGUI output

1 cd ../task03-CHARMMGUI-membrane
2 tar -xzvf charmm-gui.tgz
3 cd charmm-gui/gromacs
4 sed -i ’s:gmx d:gmx:g’ README
This step is not really needed if you
have installed the double precision
version of Gromacs which I
recommend for minimization
5 csh README

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 How to use gromacs? Task03: Membrane simulation using CHARMMGUI

Analyzing membrane simulation

Concatenating trajectories
1 gmx trjcat -f step7*.trr -o total.xtc (You can remove the *.trr files that occupy several GB)

Density profiles along the axis perpendicular membrane plane

1 echo ”0 0 1 2 3 4 5 6 7 8 9” | gmx density -f total.xtc -s step7 1.tpr -o dens.xvg -center
-d Z -ng 10
2 xmgrace -free -nxy dens.xvg

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 How to use gromacs? Task04: Simulation of a transmembrane protein using Martini

Task04: Simulation of a membrane protein using Martini

Purpose of the task

Show how tools can simplify the
execution workflow of MD
simulations
Show a more biologically
relevant system
Show a coarse grained model for
biological systems

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 How to use gromacs? Task04: Simulation of a transmembrane protein using Martini

Create a membrane protein using martinize.py

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 Appendixes Commands cheatsheet

Appendix1: Important commands

Gromacs
gmx command -h
Prepare simulation files:
gmx pdb2gmx
(protein itp file form pdb)
gmx grompp xmgrace: (Plotting xvg files)
(tpr file from top, gro and mdp) xmgrace -free -nxy XVGFILE
Run simulation:
gmx mdrun -v -deffnm TPRFILE vmd: (Vissualizing trajectories
(run MD from tpr file) vmd GROFILE XTCFILE WHOLE
Post-processing trajectory:
gmx energy -f EDRFILE
(Output trajectories observables)
gmx trjconv
(process trajectory for analysis)
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Hector Martinez-Seara, Lukasz Cwiklik Intro Gromacs 5 29/11/2017 80 / 80