Ribonuclease A is a digestive enzyme, secreted by the pancreas. The enzyme can be cleaved by subtilisin at a single peptide bond to yield Ribonuclease-S, a catalytically active complex of an S-peptide moiety (residues 1-20) and an S-protein moiety (residues 21-124), bound together by multiple non-covalent links (Stryer, 1988).
The S-Peptide has been studied in many ways, experimentally as well as theoretically (simulation) because of the high a-helix content in solution, which is remarkable in such a small peptide.
All the files of speptide are stored in the directory tutor/speptide. First go to this directory:cd speptide
To be able to simulate the S-Peptide we need a starting structure. This can
be taken from the protein data bank. There are a number of different
structure for Ribonuclease S, from one of which we have cut out the
first 20 residues, and stored it in
Have a look at the file
If you have access to a molecular
graphics program such as rasmol,
you can look at the molecule on screen, eg:
The following steps have to be taken to perform a simulation of the peptide.
We will describe in detail how such a simulation can be done, starting from a pdb-file.
Generate a molecular topology and a structure file in
format. This can be done with the pdb2gmx program:
|pdb2gmx -f speptide.pdb -p speptide.top -o speptide.gro|
Note that the correct file extension are added automatically to the
filenames on the command line.
You will only be asked to choose a forcefield, choose 0, but you can also
have pdb2gmx ask you
about protonation of residues, and about protonation of N- and C-terminus.
You can type
to see the available options.
pdb2gmx program has generated a topology file
speptide.top and a
GROMACS structure file speptide.gro and it will
positions. The -p and -o options with he
filenames are optional; without them the files topol.top and
conf.gro will be generated.
Now have a look at the output from pdb2gmx,
You will see a close resemblance to the pdb file, only the layout of
the file is a bit different.
Also do have a look at the topology
You will see a large file containing the atom types, the physical bonds between atoms, etcetera.
This is done using the programs
will make a rectangular box with empty space of user specified size
around the molecule.
will read the structure file and fill the box with water.
editconf -f speptide -o -d 0.5|
genbox -cp out -cs -p speptide -o b4em
The program prints some lines of user information, like the volume of
the box and the number of water molecules added to your
also changes the topology file
speptide.top to include
these water molecules in the topology. This can been seen by looking
at the bottom of the
You will see some lines like
[ system ] ; Name Number Protein 1 SOL N
where N is the number of water molecules added to your system by genbox.
It is also possible to solvate a peptide in another solvent such as dimethylsulfoxide (DMSO), as has been done by Mierke & Kessler, 1991.
By default, most GROMACS programs generate a set of index groups to select
the most common subsets of atoms from your system (e.g. Protein, Backbone,
C-alpha's, Solute, etc.).
For the special cases when you need to select other groups than the
default ones, an index file
can be generated using make_ndx.
This is an interactive program that lets you manipulate molecules,
residues and atom. It's use should be self-explanatory. To invoke the
program you would
|make_ndx -f b4em|
but don't bother for now.
Now we have to perform an energy minimization of the
structure to remove the local strain in the peptide (due to generation
of hydrogen positions) and to remove bad Van der Waals contacts
(particles that are too close). This can be done with the
mdrun program which
is the MD and EM program. Before we can use the
however, we have to preprocess the topology file (
structure file (
speptide.gro) and a
special parameter file (em.mdp). Check
the contents of this file
Preprocessing is done with the preprocessor called
grompp. This reads
up the files just mentioned:
|grompp -v -f em -c b4em -o em -p speptide|
In this command the -v option turns on verbose mode, which
gives a little bit of clarifying info on what the program is doing.
We now have made a run input file (em.tpr) which
serves as input for the
mdrun program. Now
we can do the energy minimization:
|mdrun -v -s em -o em -c after_em -g emlog|
In this command the -v option turns on verbose mode again. The -o option sets the filename for the trajectory file, which is not very important in energy minimizations. The -c option sets the filename of the structure file after energy minimization. This file we will subsequently use as input for the MD run. The energy minimization takes some time, the amount depending on the CPU in your computer, the load of your computer, etc. The mdrun program is automatically niced; it runs at low priority. All programs that do extensive computations are automatically run at low priority. For most modern workstations this computation should be a matter of minutes. The minimization is finished when either the minimization has converged or a fixed number of steps has been performed. Since the system consists merely of water, a quick check on the potential energy should reveal whether the minimization was successful: the potential energy of 1 SPC water molecule at 300 K is -42 kJ mole-1. Since we have about 2.55e+03 SPC molecules the potential energy should be about -1.1e+5 kJ mol-1. If the potential energy after minimization is lower than -1.1e+05 kJ mol-1 it is acceptable and the structure can be used for MD calculations. After our EM calculation the program prints something like:
STEEPEST DESCENTS converged to 2000 Potential Energy = -1.19482e+05
which means our criterium is met, and we can proceed to the next step.
Position restrained MD means Molecular Dynamics in which a part of the system is not allowed to move far off their starting positions. To be able to run with position restraints we must add a section to the speptide.top file, describing which atoms are to be restrained. Such a section is actually generated by the pdb2gmx program. In the topology file it looks like
In the topology file we use conditional inclusion, i.e. only if a variable POSRES is set in the preprocessor do we include the file, this allows us to use the same topology file for runs with and without position restraints. In the pr.mdp parameter file for the position restraints this variable is set indeed:
define = -DPOSRES
At last we can generate the input for the position restrained mdrun:
|grompp -f pr -o pr -c after_em -r after_em -p speptide|
Now it's MDrun time:
|mdrun -v -s pr -e pr -o pr -c after_pr -g prlog >& pr.job &|
This run is started in the background (it will take a while), you
can watch how long it will take by typing:
|tail -f pr.job|
With the Ctrl-C key you can kill the tail command.
A good check of your simulation is to see whether density and potential
energies have converged:
|g_energy -f pr -o out -w|
g_energy program will prompt you to select a number of energy terms
from a list. For potential energy type:
If you have the xmgr program installed it will automatically pop up on your screen with the energy plot. You can do the same for the density and other energy terms, such as Solvent-Protein interactions.
Full MD is very similar to the restrained MD as far as GROMACS is
concerned. Check out the full.mdp for details.
|grompp -v -f full -o full -c after_pr -p speptide|
Then we can start mdrunning
|mdrun -v -s full -e full -o full -c after_full -g flog >& full.job &|
You should do similar convergence checks (and more!) as for the position
We will not describe analysis in detail, because most analysis tools are described in the Analysis chapter of the printed manual. We just list a few of the possibilities within GROMACS. By now you should be able to start programs yourself.
View the trajectory on your own X-screen (program
|ngmx -s pr -f full|
The Root Mean Square Deviation (RMSD) with respect to the crystal
g_rms) is a measure of how well the
crystal (starting) structure is maintained in the simulation.
The RMSD at time t is computed as
after performing a least squares super position of the structure at
time t onto the structure at time 0.
|g_rms -s pr -f full -o rmsd|
The Radius of Gyration (Rg, program
g_gyrate)) is a measure of the size of the
protein. It is computed as the mean square distance of atoms from the center
of mass of the molecule: Rg(t) = SUMi=1 .. N
|g_gyrate -s pr -f full -o gyrate|
The Ramachandran Plot shows whether the backbone torsion angles
(φ/ψ) of your
peptide are within the allowed region.
|g_rama -s pr -f full -o rama|
A salt bridge analysis (program
g_saltbr) will tell you whether
there are any saltbridges formed or broken during the simulation.
It will also tell you about repulsive electrostatic interactions.
|g_saltbr -s pr -f full -t 0.5 -sep|
|xmgrace sb-GLU2:ARG10.xvg sb-GLU2:LYSH7.xvg -legend load|
|my_dssp -s pr -f full|
|xpm2ps -f ss.xpm -o ss.eps|
You have been witness of a full MD simulation starting from a pdb-file. It's that easy, but then again, maybe it was not that easy. The example presented here is a real example, this is how a production run should be performed, the complexity is in the process itself and not in the software (at least, that's our opinion).