Terminology

Pressure
The pressure in molecular dynamics can be computed from the kinetic energy and the virial.

Fluctuation
Whether or not pressure coupling is used within a simulation, the pressure value for the
simulation box will oscillate significantly. Instantaneous pressure is meaningless, and not well-
defined. Over a picosecond time scale it usually will not be a good indicator of the true pressure.
This variation is entirely normal due to the fact that pressure is a macroscopic property and can
only be measured properly as time average, while it is being measured and/or adjusted with
pressure coupling on the microscopic scale. How much it varies and the speed at which it does
depends on the number of atoms in the system, the type of pressure coupling used and the
value of the coupling constants. Fluctuations of the order of hundreds of bar are typical. For a
box of 216 waters, fluctuations of 500-600 bar are standard. Since the fluctuations go down with
the square root of the number of particles, a system of 21600 water molecules (100 times larger)
will still have pressure fluctuations of 50-60 bar.

Periodic boundary conditions

Periodic boundary conditions (PBC) are used in molecular dynamics simulations to avoid
problems with boundary effects caused by finite size, and make the system more like an infinite
one, at the cost of possible periodicity effects.

Beginners visualizing a trajectory sometimes think they are observing a problem when

the molecule(s) does not stay in the centre of the box, or

it appears that (parts of) the molecule(s) diffuse out of the box, or
holes are created, or
broken molecules appear, or
their unit cell was a rhombic dodecahedron or cubic octahedron but it looks like a slanted cube
after the simulation, or
crazy bonds all across the simulation cell appear.

This is not a problem or error that is occurring, it is what you should expect.

The existence of PBC means that any atom that leaves a simulation box by, say, the right-hand
face, then enters the simulation box by the left-hand face. In the example of a large protein, if
you look at the face of the simulation box that is opposite to the one from which the protein is
protruding, then a hole in the solvent will be visible. The reason that the molecule(s) move from
where they were initially located within the box is (for the vast majority of simulations) they are
free to diffuse around. And so they do. They are not held in a magic location of the box. The box
is not centered around anything while performing the simulation. Molecules are not made whole
as a matter of course. Moreover, any periodic cell shape can be expressed as a parallelepiped
(a.k.a. triclinic cell), and GROMACS does so internally regardless of the initial shape of the box.

These visual issues can be fixed after the conclusion of the simulation by judicious use of the
optional inputs to gmx trjconv to process the trajectory files. Similarly, analyses such as RMSD of
atomic positions can be flawed when a reference structure is compared with a structure that
needs adjusting for periodicity effects, and the solution with gmx trjconv follows the same lines.
Some complex cases needing more than one operation will require more than one invocation of
gmx trjconv in order to work.

For further information, see the corresponding section in the Reference Manual.

Suggested workflow
Fixing periodicity effects with gmx trjconv to suit visualization or analysis can be tricky. Multiple
invocations can be necessary. You may need to create custom index groups (e.g. to keep your
ligand with your protein) Following the steps below in order (omitting those not required) should
help get a pleasant result. You will need to consult gmx trjconv -h to find out the details for
each step. That’s deliberate – there is no magic “do what I want” recipe. You have to decide what
you want, first. :-)

1. First make your molecules whole if you want them whole.

2. Cluster your molecules/particles if you want them clustered.
3. If you want jumps removed, extract the first frame from the trajectory to use as the reference,
and then use -pbc nojump with that first frame as reference.
4. Center your system using some criterion. Doing so shifts the system, so don’t use -pbc
nojump after this step.

5. Perhaps put everything in some box with the other -pbc or -ur options.
6. Fit the resulting trajectory to some (other) reference structure (if desired), and don’t use any
PBC related option afterwards.

With point three, the issue is that gmx trjconv removes the jumps from the first frame using the
reference structure provided with -s. If the reference structure (run input file) is not
clustered/whole, using -pbc nojump will undo steps 1 and 2.

Thermostats
Thermostats are designed to help a simulation sample from the correct ensemble (i.e. NVT or
NPT) by modulating the temperature of the system in some fashion. First, we need to establish
what we mean by temperature. In simulations, the “instantaneous (kinetic) temperature” is
usually computed from the kinetic energy of the system using the equipartition theorem. In other
words, the temperature is computed from the system’s total kinetic energy.
So, what’s the goal of a thermostat? Actually, it turns out the goal is not to keep the temperature
constant, as that would mean fixing the total kinetic energy, which would be silly and not the aim
of NVT or NPT. Rather, it’s to ensure that the average temperature of a system be correct.

To see why this is the case, imagine a glass of water sitting in a room. Suppose you can look
very closely at a few molecules in some small region of the glass, and measure their kinetic
energies. You would not expect the kinetic energy of this small number of particles to remain
precisely constant; rather, you’d expect fluctuations in the kinetic energy due to the small number
of particles. As you average over larger and larger numbers of particles, the fluctuations in the
average get smaller and smaller, so finally by the time you look at the whole glass, you say it has
“constant temperature”.

Molecular dynamics simulations are often fairly small compared to a glass of water, so we have
bigger fluctuations. So it’s really more appropriate here to think of the role of a thermostat as
ensuring that we have

a. the correct average temperature, and

b. the fluctuations of the correct size.

See the relevant section in the Reference Manual for details on how temperature coupling is
applied and the types currently available.

What to do
Some hints on practices that generally are a good idea:

Preferably, use a thermostat that samples the correct distribution of temperatures (for
examples, see the corresponding manual section), in addition to giving you the correct
average temperature.
At least: use a thermostat that gives you the correct average temperature, and apply it to
components of your system for which they are justified (see the first bullet in What not to do).
In some cases, using tc-grps = System may lead to the “hot solvent/cold solute” problem
described in the 3rd reference in Further reading.

What not to do
Some hints on practices that generally not a good idea to use:

Do not use separate thermostats for every component of your system. Some molecular
dynamics thermostats only work well in the thermodynamic limit. A group must be of sufficient
size to justify its own thermostat. If you use one thermostat for, say, a small molecule, another
for protein, and another for water, you are likely introducing errors and artifacts that are hard
to predict. In particular, do not couple ions in aqueous solvent in a separate group from that
solvent. For a protein simulation, using tc-grps = Protein Non-Protein is usually best.
Do not use thermostats that work well only in the limit of a large number of degrees of
freedom for systems with few degrees of freedom. For example, do not use Nosé-Hoover or
Berendsen thermostats for types of free energy calculations where you will have a component
 of the system with very few degrees of freedom in an end state (i.e. a noninteracting small
molecule).

Further reading
1. Cheng, A. & Merz, K. M. Application of the Nosé-Hoover chain algorithm to the study of
protein dynamics. J. Phys. Chem. 100 (5), 1927–1937 (1996).
2. Mor, A., Ziv, G. & Levy, Y. Simulations of proteins with inhomogeneous degrees of freedom:
the effect of thermostats. J. Comput. Chem. 29 (12), 1992–1998 (2008).
3. Lingenheil, M., Denschlag, R., Reichold, R. & Tavan, P. The “hot-solvent/cold-solute” problem
revisited. J. Chem. Theory Comput. 4 (8), 1293–1306 (2008).

Energy conservation
In principle, a molecular dynamics simulation should conserve the total energy, the total
momentum and (in a non-periodic system) the total angular momentum. A number of algorithmic
and numerical issues make that this is not always the case:

Cut-off treatment and/or long-range electrostatics treatment (see Van Der Spoel, D. & van
Maaren, P. J. The origin of layer structure artifacts in simulations of liquid water. J. Chem.
Theor. Comp. 2, 1–11 (2006).)
Treatment of pair lists,
Constraint algorithms (see e.g. Hess, B. P-LINCS: A parallel linear constraint solver for
molecular simulation. J. Chem. Theor. Comp. 4, 116–122 (2008).).
The integration timestep.
Temperature coupling and pressure coupling.
Round-off error (in particular in single precision), for example subtracting large numbers
(Lippert, R. A. et al. A common, avoidable source of error in molecular dynamics integrators. J.
Chem. Phys. 126, 046101 (2007).).
The choice of the integration algorithm (in GROMACS this is normally leap-frog).
Removal of center of mass motion: when doing this in more than one group the conservation
of energy will be violated.

Average structure
Various GROMACS utilities can compute average structures. Presumably the idea for this comes
from something like an ensemble-average NMR structure. In some cases, it makes sense to
calculate an average structure (as a step on the way to calculating root-mean-squared
fluctuations (RMSF), for example, one needs the average position of all of the atoms).

However, it’s important to remember that an average structure isn’t necessarily meaningful. By
way of analogy, suppose I alternate holding a ball in my left hand, then in my right hand. What’s
the average position of the ball? Halfway in between – even though I always have it either in my
left hand or my right hand. Similarly, for structures, averages will tend to be meaningless anytime
there are separate metastable conformational states. This can happen on a sidechain level, or
for some regions of backbone, or even whole helices or components of the secondary structure.

Thus, if you derive an average structure from a molecular dynamics simulation, and find artifacts
like unphysical bond lengths, weird structures, etc., this doesn’t necessarily mean something is
wrong. It just shows the above: an average structure from a simulation is not necessarily a
physically meaningful structure.

Blowing up
Blowing up is a highly technical term used to describe a common sort of simulation failure. In
brief, it describes a failure typically due to an unacceptably large force that ends up resulting in a
failure of the integrator.

To give a bit more background, it’s important to remember that molecular dynamics numerically
integrates Newton’s equations of motion by taking small, discrete timesteps, and using these
timesteps to determine new velocities and positions from velocities, positions, and forces at the
previous timestep. If forces become too large at one timestep, this can result in extremely large
changes in velocity/position when going to the next timestep. Typically, this will result in a
cascade of errors: one atom experiences a very large force one timestep, and thus goes
shooting across the system in an uncontrolled way in the next timestep, overshooting its
preferred location or landing on top of another atom or something similar. This then results in
even larger forces the next timestep, more uncontrolled motions, and so on. Ultimately, this will
cause the simulation package to crash in some way, since it can’t cope with such situations. In
simulations with constraints, the first symptom of this will usually be some LINCS or SHAKE
warning or error – not because the constraints are the source of the problem, but just because
they’re the first thing to crash. Similarly, in simulations with domain decomposition, you may see
messages about particles being more than a cell length out of the domain decomposition cell of
their charge group, which are symptomatic of your underlying problem, and not the domain
decomposition algorithm itself. Likewise for warnings about tabulated or 1-4 interactions being
outside the distance supported by the table. This can happen on one computer system while
another resulted in a stable simulation because of the impossibility of numerical reproducibility of
these calculations on different computer systems.

Possible causes include:

you didn’t minimize well enough,

you have a bad starting structure, perhaps with steric clashes,
you are using too large a timestep (particularly given your choice of constraints),
you are doing particle insertion in free energy calculations without using soft core,
you are using inappropriate pressure coupling (e.g. when you are not in equilibrium,
Berendsen can be best while relaxing the volume, but you will need to switch to a more
accurate pressure-coupling algorithm later),
 you are using inappropriate temperature coupling, perhaps on inappropriate groups, or
your position restraints are to coordinates too different from those present in the system, or
you have a single water molecule somewhere within the system that is isolated from the other
water molecules, or
you are experiencing a bug in gmx mdrun.

Because blowing up is due, typically, to forces that are too large for a particular timestep size,
there are a couple of basic solutions:

make sure the forces don’t get that large, or

use a smaller timestep.

Better system preparation is a way to make sure that forces don’t get large, if the problems are
occurring near the beginning of a simulation.

Diagnosing an unstable system

Troubleshooting a system that is blowing up can be challenging, especially for an inexperienced
user. Here are a few general tips that one may find useful when addressing such a scenario:

1. If the crash is happening relatively early (within a few steps), set nstxout (or nstxout-
compressed ) to 1, capturing all possible frames. Watch the resulting trajectory to see which
atoms/residues/molecules become unstable first.
2. Simplify the problem to try to establish a cause:
If you have a new box of solvent, try minimizing and simulating a single molecule to see if
the instability is due to some inherent problem with the molecule’s topology or if instead
there are clashes in your starting configuration.
If you have a protein-ligand system, try simulating the protein alone in the desired solvent. If
it is stable, simulate the ligand in vacuo to see if its topology gives stable configurations,
energies, etc.
Remove the use of fancy algorithms, particularly if you haven’t equilibrated thoroughly first
3. Monitor various components of the system’s energy using gmx energy. If an intramolecular
term is spiking, that may indicate improper bonded parameters, for example.
4. Make sure you haven’t been ignoring error messages (missing atoms when running gmx
pdb2gmx, mismatching names when running gmx grompp, etc.) or using work-arounds (like
using gmx grompp -maxwarn when you shouldn’t be) to make sure your topology is intact and
being interpreted correctly.
5. Make sure you are using appropriate settings in your mdp file for the force field you have
chosen and the type of system you have. Particularly important settings are treatment of
cutoffs, proper neighbor searching interval ( nstlist ), and temperature coupling. Improper
settings can lead to a breakdown in the model physics, even if the starting configuration of the
system is reasonable.
When using no explict solvent, starting your equilibration with a smaller time step than your
production run can help energy equipartition more stably.

There are several common situations in which instability frequently arises, usually in the
introduction of new species (ligands or other molecules) into the system. To determine the
source of the problem, simplify the system (e.g. the case of a protein-ligand complex) in the
following way.

1. Does the protein (in water) minimize adequately by itself? This is a test of the integrity of the
coordinates and system preparation. If this fails, something probably went wrong when
running gmx pdb2gmx (see below), or maybe gmx genion placed an ion very close to the
protein (it is random, after all).
2. Does the ligand minimize in vacuo? This is a test of the topology. If it does not, check your
parameterization of the ligand and any implementation of new parameters in force field files.
3. (If previous item is successful) Does the ligand minimize in water, and/or does a short
simulation of the ligand in water succeed?

Other sources of possible problems are in the biomolecule topology itself.

1. Did you use -missing when running gmx pdb2gmx? If so, don’t. Reconstruct missing
coordinates rather than ignoring them.
2. Did you override long/short bond warnings by changing the lengths? If so, don’t. You probably
have missing atoms or some terrible input geometry.

Molecular dynamics
Molecular dynamics (MD) is computer simulation with atoms and/or molecules interacting using
some basic laws of physics. The GROMACS Reference Manual provides a good general
introduction to this area, as well as specific material for use with GROMACS. The first few
chapters are mandatory reading for anybody wishing to use GROMACS and not waste time.

Introduction to molecular modeling (slides, video)] - theoretical framework, modeling levels,

limitations and possibilities, systems and methods (Erik Lindahl).

Books
There are several text books around.

Good introductory books are:

A. Leach (2001) Molecular Modeling: Principles and Applications.

T. Schlick (2002) Molecular Modeling and Simulation

With programming background:

D. Rapaport (1996) The Art of Molecular Dynamics Simulation

 D. Frenkel, B. Smith (2001) Understanding Molecular Simulation

More from the physicist’s view:

M. Allen, D. Tildesley (1989) Computer simulation of liquids

H.J.C. Berendsen (2007) Simulating the Physical World: Hierarchical Modeling from Quantum
Mechanics to Fluid Dynamics

Types / Ensembles
NVE - number of particles (N), system volume (V) and energy (E) are constant / conserved.
NVT - number of particles (N), system volume (V) and temperature (T) are constant /
conserved. (See thermostats for more on constant temperature).
NPT - number of particles (N), system pressure (P) and temperature (T) are constant /
conserved. (See pressure coupling for more on constant pressure).

Force field
Force fields are sets of potential functions and parametrized interactions that can be used to
study physical systems. A general introduction to their history, function and use is beyond the
scope of this guide, and the user is asked to consult either the relevant literature or try to start at
the relevant Wikipedia page.

Copyright © 2024, GROMACS development team

Made with Sphinx and @pradyunsg's Furo