Methods 41 (2007) 475488

www.elsevier.com/locate/ymeth

Setting up and running molecular dynamics simulations

of membrane proteins
Christian Kandt, Walter L. Ash, D. Peter Tieleman
Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary AB, Canada T2N 1N4

Accepted 17 August 2006

Abstract

Molecular dynamics simulations have become a popular and powerful technique to study lipids and membrane proteins. We present
some general questions and issues that should be considered prior to embarking on molecular dynamics simulation studies of membrane
proteins and review common simulation methods. We suggest a practical approach to setting up and running simulations of membrane
proteins, and introduce two new (related) methods to embed a protein in a lipid bilayer. Both methods rely on placing lipids and the pro-
tein(s) on a widely spaced grid and then shrinking the grid until the bilayer with the protein has the desired density, with lipids neatly
packed around the protein. When starting from a grid based on a single lipid structure, or several potentially diVerent lipid structures
(method 1), the bilayer will start well-packed but requires more equilibration. When starting from a pre-equilibrated bilayer, either pure
or mixed, most of the structure of the bilayer stays intact, reducing equilibration time (method 2). The main advantages of these methods
are that they minimize equilibration time and can be almost completely automated, nearly eliminating one time consuming step in MD
simulations of membrane proteins.
2006 Elsevier Inc. All rights reserved.

Keywords: Molecular dynamics simulation; Membrane proteins; Molecular modeling; Electrostatics; Algorithms

1. Introduction about membrane proteins: to date the 3D structures of 97

1.1. Membrane proteins
A fundamental precondition for life is the compartmen-
talization of cells and organelles from their environment
that is provided by biological membranes. Beyond this
basic role as a barrier, biomembranes facilitate a number of
other functions that are mainly determined by the type of
proteins associated with or embedded in the bilayer. Mem-
brane proteins are therefore of high biological relevance, as
they are key players in crucial processes such as energy con-
version, transport, signal recognition, and transduction.
Compared to soluble proteins it is particularly diYcult
to obtain high-resolution structural experimental data
*
Corresponding author. Fax: +1 403 289 9311.
E-mail address: tieleman@ucalgary.ca (D. Peter Tieleman).
diVerent membrane proteins have been solved, whereas
about 13,000 structures are available for soluble proteins
[1]. Gaining a deeper insight into the architecture of mem-
brane proteins is thus clearly a major goal in modern struc-
tural biology. Apart from further development and
improvement of structure resolving experimental tech-
niques, theoretical and computational methods have
become increasingly important. These range from bioinfor-
matics and homology modeling procedures [24] to quan-
tum mechanical calculations and molecular mechanical
simulations [512].
1.2. Molecular dynamics simulations
Molecular dynamics (MD) simulations numerically
investigate the motions of a system of discrete particles
under the inXuence of internal and external forces. The
spectra of possible applications based on this approach is

1046-2023/$ - see front matter 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.ymeth.2006.08.006
476 C. Kandt et al. / Methods 41 (2007) 475488
broad, ranging from atoms in a molecule to stars in a gal-
axy [13]. However, the underlying principles are the same:
interactions of the respective particles are empirically
described by a potential energy function from which the
forces that act on each particle are derived. With knowledge
of these forces it is possible to calculate the dynamic behav-
ior of the system using classical equations of motion, in
their simplest form Newtons law, for all atoms in the sys-
tem. For biomolecular systems, a discrete time step of up to
a few femtoseconds is used, with typical simulations
consisting of millions of steps.
For an atomic system, the potential energy function con-
sists of a set of equations that empirically describe bonded
and non-bonded interactions between atoms. This energy
function together with the set of its empirical parameters is
referred to as the force Weld. Molecular dynamics force
Welds usually consist of two major components. The Wrst
part describes interactions between atoms connected via
covalent bonds, which typically includes bonds, bond
angles, and dihedrals. The second part treats non-bonded
interactions, typically as electrostatic interactions between
the (partial) charges on each atom and a Lennard-Jones
potential to model dispersive van der Waals interactions.
Each molecule in the simulation is described by its topol-
ogy, the combination of the set of all atoms with their
non-bonded and bonded interaction parameters and the
connectivity of those atoms in the molecule.
Since their introduction in the late 1950s [14,15] and
their Wrst application to a protein [16], MD simulations
have become a common tool to investigate structureactiv-
ity relationships in biological macromolecules. Providing
the element of dynamics at an atomic level of detail, these
simulations facilitate the interpretation of experimental
data and give access to information not directly accessible
by experiments. For biomolecular MD simulations, systems
of more than 100,000 atoms simulated on a time scale of
nanoseconds have become standard. MD simulations also
form a critical component of atomic-resolution structure
determination methods such as X-ray crystallography and
NMR. For a more detailed introduction to MD simula-
tions see for example [13,1719].
1.3. MD simulation of membrane proteins
Since the Wrst simulation of a membrane-embedded pep-
tide in 1994 [20] and an integral membrane protein in 1995
[21] molecular dynamics simulations of membrane proteins
have come a long way. To date, successfully investigated
systems include interface-associated and trans-membrane
peptides, fusion proteins, channel and pore proteins, trans-
porters, ion pumps, ATP-synthases and G-protein coupled
receptors. Recent overviews of the Weld can be found in
[22,23].
In this paper we review the most commonly used meth-
ods to set up molecular dynamics simulations of membrane
proteins and suggest a practical approach to setting up and
running such simulations. We introduce two new and
eYcient techniques to generate a starting structure and also
discuss some general questions and issues that should be
considered prior to any modeling or simulation. In our
group we mainly use GROMACS [24,25], a popular, freely
available, and relatively user-friendly set of programs for
MD simulations. At the time of writing this paper, the cur-
rent version is 3.3.1. Although some of the details will be
diVerent for other software packages, and for other ver-
sions of GROMACS, the steps and algorithms we describe
will be similar and can be readily implemented.
2. Description of methods
2.1. Simulation at all?
When deciding to approach a speciWc problem in mem-
brane protein biology using simulations, it is worthwhile to
reXect on some primary questions prior to any modeling or
simulation. Is the problem to be investigated accessible by
MD at all? Are the simulations practical or likely to deliver
interesting and informative results? For large membrane
protein systems with a total size of 100,000 atoms or more,
simulation times on the scale of tens of nanoseconds are
currently accessible using advanced supercomputers. Is this
time scale enough for the particular system of interest? On
what time scales do the phenomena of interest take place?
Are current simulations accurate enough? Small diVerences
in binding free energies of the order of 1 kT are not likely to
be accurately reproduced in simulations, even if there are
no issues with insuYcient sampling. Is there further experi-
mental data available that can be used for validating simu-
lation results? At times, the answer to these questions
should suggest that simulation is not an appropriate
method to address the problem at hand.
If simulation seems an appropriate approach, additional
questions arise. Are appropriate starting structures avail-
able? Do computational models of the lipids of interest
exist, or do they have to be developed (useful, but a sub-
stantial eVort)? Are unbiased equilibrium MD simulations
an appropriate choice or would non-equilibrium techniques
be more suitable? For example, large scale conformational
changes can be forced to take place on an accessible time
scale using controlled MD techniques [26,27] or by per-
forming pulling experiments [28], although both methods
also introduce artifacts that need to be evaluated. Free
energy-related properties can rarely be determined from
equilibrium simulations of complex systems, but specialized
methods, such as umbrella sampling, can sometimes be
used [29,30]. Is there a parameter set that adequately
describes the lipids and protein of interest, and perhaps
small-molecule ligands? Many membrane proteins are sen-
sitive to the lipid environment, and thus may be critically
aVected by the membrane model used [31]. Are there
parameters missing that have to be determined Wrst? Are
topologies available for all of the systems components?
Which software should be used? This depends on the prob-
lem at hand, the availability of local expertise, computa-
 C. Kandt et al. / Methods 41 (2007) 475488
tional facilities, and other factors. Is enough computer time
available for the planned simulations? Below we address
some of the technical questions, but there is no general
answer for the suitability of simulations for a particular
problem.
2.2. Force Welds
2.2.1. Overview
Force Welds could be considered the primary assumption
in MD simulationsthey describe the interaction between
atoms. There are many diVerent force Welds [32], but cur-
rently there are only four widely used force Welds for simu-
lating biological macromolecules: Amber [33,34],
CHARMM [35,36], GROMOS [37] and OPLS [38]. Each of
these has undergone continuous development as parame-
terization methodology and experimental techniques
advance, and to correctly specify a force Weld an exact revi-
sion number is required. All four force Welds reproduce
many protein characteristics satisfyingly well [32,39]. They
have some diVerent strengths and weaknesses, but most
importantly they share a number of the same limitations
due to the basic simplifying assumptions necessary in large
scale MD simulations. The treatment of electrostatics in
particular is somewhat simplistic because electronic polar-
izability is only accounted for in an average way. Force
Weld and methods development aimed at resolving these
deWciencies is ongoing [32,3941].
Phospholipids are a subset of biomolecules that have
received considerably less attention than proteins or nucleic
acids. There are only two phospholipid force Welds in com-
mon use today, although additional sets are in development
(e.g. [42]). The Wrst commonly used lipid force Weld is part
of CHARMM [43,44]; the second is based on an older ver-
sion of OPLS and AMBER with some additional parame-
ters from Berger et al. [45]. Both force Welds are able to
reproduce the available information from stable bilayers
fairly well, although both sets of lipid parameters will ben-
eWt from further reWnements. Recent examples are the treat-
ment of the rotational isomerization of acyl tails, where
intermediate-range intramolecular interactions play an
important role [46], and a direct comparison of simulations
of lipids with diVraction experiments [47].
Unlike CHARMM lipids and most of the recent protein
force Welds, Berger lipids are described by a united-atom
force Weld; each aliphatic carbon with associated hydrogens
is described by a single particle with the approximate physi-
cal characteristics of a methyl, methylene, or methine
group. This is a level of detail somewhere between fully
atomistic descriptions and some of the coarse-grained
approaches that have been developed over the years [48,49].
When hydrogens are treated explicitly, the number of
atoms per lipid approximately triples, and the number of
pairwise interactions in the membrane becomes much
higher. Because the computationally inexpensive united-
atom lipids reproduce experimental behavior reasonably
well, it may be desirable to use a combination of united-
477
atom lipids and an accurate all-atom protein force Weld [50].
Ultimately, an all-atom force Weld should be more accurate,
but despite some discussion in the literature we do not
believe that we have reached the point yet where the united-
atom approximation is the limiting factor in accuracy for
most purposes.
This point is related to a broader problem. SigniWcant
progress has been made in developing modern protein force
Welds, based on a comparison with high-level quantum
mechanics and high-resolution experiments on soluble pro-
teins, but it is challenging to obtain accurate experimental
data to validate and improve lipid models. The situation is
even more complex for lipidprotein interactions compared
to pure lipids, because experiments on membrane proteins
and peptides typically have a signiWcantly lower resolution
than in solution. In addition, from a simulation point of
view, critical tests are diYcult due to the long simulations
required. For now, the most straightforward approach to
membrane protein simulations involves directly combining
mathematically compatible protein and lipid force Welds,
and this approach has yielded useful insights into
membrane protein behavior [22,23].
A key diYculty with classical simulations of molecules
that move between two very diVerent environments is that
the parameters used to describe them have to be accurate in
both environments, although typical biomolecular force
Welds have been developed for aqueous solution. A direct
approach to testing the free energies of transfer between
water and hydrophobic environments is now possible com-
putationally [5054], which allows a direct comparison to
experimentally measured partition coeYcients [55] and the
interfacial partitioning of model peptides [56]. Such studies
have found substantial errors in free energies of transfer for
amino acid side chain analogues between water and cyclo-
hexane. SigniWcant improvements in current force Welds can
be expected based on re-parameterization eVorts, both in
the thermodynamics of membrane proteins at the water/
lipid interface and in processes such as small molecules
binding to proteins, which typically also involves a change
of environment. There will be a limit to the maximum accu-
racy that can be reached by simple re-parameterization due
to ignoring electronic polarizability eVects (e.g. [40]), but in
our opinion we have not reached the point yet where fur-
ther improvement is impossible without including such
eVects. An intriguing question is whether a general polariz-
able force Weld would solve many problems at once, possi-
bly with ultimately less eVort. Undoubtedly we will see
many endeavors in the near future aimed at improving
protein and lipid force Welds.
2.3. Building topologies
Membrane proteins are often associated with speciWc
cofactors, ligands or other biological molecules for which
the simulation package holds no topologies. In many cases,
such descriptions might already exist in another force Weld
and might be converted; in other cases, they must be built
478 C. Kandt et al. / Methods 41 (2007) 475488
anew. However, care must be taken to ensure their accuracy
and compatibility with the protein and lipid models. A use-
ful and commonly used approach when generating a new
topology is to dissect the molecule into more manageable
parts. For example, complex substrates and cofactors often
contain amino acid-like fragments for which force Weld
parameters are already available, which thus require only
slight adjustments. High-resolution X-ray data can be used
for reference when parameterizing bond length and angle
deWnitions. Force constants for bonds and angles can in
principle be obtained from accurate QM calculations, but
in practice bonds are often taken as Wxed (so that only the
bond length matters) and angle force constants are chosen
similar to existing angle parameters in the force Weld.
One important factor to consider when evaluating a
model for a cofactor is its partial charge distribution, or
more generally, the way the force Weld models non-bonded
interactions. Point charges and Leonard-Jones interactions
that give the proper behavior for a molecule in water may
not be adequate to describe behavior in the interior of a
bilayer or a hydrophobic pocket in a protein. The charge
distribution (usually derived from ab initio QM methods)
should be compatible with the protein force Weld; a charge
partitioning technique that overestimates charges on a
cofactor relative to a protein may artiWcially and adversely
aVect the balance of forces controlling protein-cofactor
interactions. The interactions between ions and a potassium
channel are an interesting example [57]. Thus it is advisable
to use a methodology similar to that used in parameterizing
the protein (and/or lipid) force Weld.
Topologies of complex molecules present a signiWcant
practical problem. MD software typically generates topolo-
gies for biopolymers like proteins and polynucleotides
automatically from the 3D coordinates, but this relies on an
existing description (in the software) of the exact topology
of amino acids and individual nucleotides. It is a challeng-
ing problem to generate accurate topologies for arbitrary
molecules. Although there are a number of web servers
[5860] and computer programs (e.g., AMBER, Insight II)
that will attempt to generate topologies based on chemical
structures or 3D coordinates, in practice these require care-
ful manual checking and sometimes adjustments. At the
moment topologies of complex molecules like vitamin B12
require signiWcant manual eVort [61].
2.4. Creating a starting structure
In addition to addressing general questions and chosing
simulations parameters, setting up a membrane protein
simulation system usually requires four major working
steps, which will be discussed in the following sections.
These steps are: 1, orienting the protein; 2, preparation of
the bilayer; 3, solvating the system; 4, system equilibration.
As pointed out earlier, in describing these processes we
make explicit reference to GROMACS utilities [24,25] dis-
tributed at the time of writing (version 3.3.1). However the
methods described herein do not rely on speciWc software
and can be readily implemented with other molecular
dynamics and modeling programs.
2.4.1. Orienting the protein
When starting from a pre-existing bilayer the common
approach begins with centering the protein in the simula-
tion box. Both the protein and the bilayer box should have
the same dimensions so the two coordinate sets can be com-
bined easily. The protein is then oriented in such a way that
its hydrophobic belt is aligned with the non-polar lipid tails.
This can be done using either common molecular graphic
programs such as MolMol [62], VMD [63] or RasMol [64]
or via the GROMACS tool editconf, applying explicit
translation and rotational vectors to the protein only. The
term hydrophobic belt refers to a common feature of mem-
brane proteins regarding the distribution of charged resi-
dues on the molecules surface: the area exposed to the
hydrophobic component of a bilayer usually contains no
charged residues [65,66] (see Fig. 1).
For peptides the procedure is the same or even simpler
when dealing with short (20 residues) trans-membrane
peptides, where their entire length does not exceed the
bilayer thickness. When constructing the membrane around
the protein instead of starting from a pre-equilibrated
bilayer, one should ensure that lipids are placed in such a
way that the hydrophobic belt criterion is met. Further
reWnements are possible, as the hydrophobic belt criterion
is not exact and could be replaced by a more quantitative
assessment based on e.g., optimizing the electrostatic free
energy of solvation using continuum electrostatics [67].
2.4.2. Preparation of the bilayer
Many important motions exhibited by lipids and lipid
bilayers are relatively slow compared to the time scale of
MD simulations [6870], including such processes as lipid
diVusion and rotational reorientation, phase transitions,
bilayer self-assembly, and lipid Xip-Xop. Therefore, one
major diYculty in designing a membrane protein simulation
Fig. 1. (a) Membrane proteins typically exhibit a characteristic distribu-
tion of charged residues (black) on their molecular surface (white)
whereas the area exposed to the hydrophobic component of the bilayer
consists of neutral residues only (a). This hydrophobic belt (b) can be used
to align the protein with the polar lipid head groups (grey). (Figure
created using MolMol and XaraX1).
 C. Kandt et al. / Methods 41 (2007) 475488
experiment is the proper assembly of the starting lipid
bilayer and protein system. There are, in essence, two possi-
ble approaches to this problem: 1. A protein is inserted into
an existing, equilibrated bilayer. 2. A bilayer is built around
the protein to embed it in the bilayer.
We Wrst review the main methods used in the literature.
Then we introduce two new and eYcient approaches that
we recommend for new simulations: one starting from the
coordinates of a single lipid molecule and one starting from
a pre-equilibrated bilayer. Either way, if not stated other-
wise all preparation techniques initially take place in the
absence of water.
In the end it does not matter which method one uses to
make a starting structure as long as the system has been
equilibrated long enough and known experimental data are
adequately reproduced. However, the practical diVerences
between approaches can be substantial. Important criteria
to favor one method over another are the time required to
equilibrate the system, the amount of manual eVort
required, and the potential for serious artifacts due to
deformations of the bilayer caused by the wrong number of
lipids in one of the leaXets relative to the other leaXet.
2.4.2.1. Review of preparation techniques
2.4.2.1.1. Delete lipids within a cut-oV. This is the sim-
plest and most commonly used method of preparing a
bilayer for membrane protein simulation (Fig. 2a). Recent
examples can be found in [7180]. The coordinate sets of
bilayer and protein have already been combined and over-
lapping lipids are removed on the basis of a simple distance
cut-oV between the protein and either single lipid atoms
(usually the phosphorus in the head groups), or the entire
lipid. The applied cut-oV length varies between 56 (based
on proteinphosphorus distances) and 0.81.6 (based on
any contact between protein atoms and whole-lipids).
The main problem with this method is that, due to the
highly disordered lipid conformations, a rather rugged hole
is created which can require a long simulation time before
the local density has returned to its equilibrium value again.
This can also lead to a shrinking of the whole membrane
patch below an area that is not suitable for the system size
anymore. The graphical representation option draw peri-
odic images in VMD oVers a particularly useful way to
check if the chosen system size is big enough to ensure the
protein does not see any of its periodic images in the
course of the simulation. It is generally useful to keep in
mind that lipid diVusion takes place on a time scale of 10
100 ns and for typical simulation studies equilibration times
of 1020 ns are necessary [68]. Non-equilibrium situations
like the presence of a hole might initially decay rapidly, but
the resulting structures may still not represent equilibrium
for a signiWcant amount of time.
Another problem frequently encountered, especially
when using the protein phosphorus approach, is the
potential for clashes between protein residues and those lip-
ids which are still present after the removal step. One possi-
ble way to deal with this is to exchange the aVected protein
479
side chains with alternative and more suitable rotamer con-
formations. This can be done using packages such as Swis-
sPDB [81,82], but we prefer to avoid this situation
altogether.
A variant of the cut-oV method uses a rectangular grid
placed on bilayer and protein. All lipids with atoms fall-
ing in grid cells already occupied by protein are removed
[83].
2.4.2.1.2. Using repulsive forces to create a hole cylinder
& mdrun_hole. The protein shape is approximated by a cyl-
inder of appropriate radius and all lipids with their phos-
phorus atoms inside this cylinder are removed (Fig. 2b).
Subsequently all lipid molecules which still have (non-phos-
phorus) atoms inside are driven out by applying a weak
repulsive potential, after which the protein can be inserted
in the generated cavity [84]. The major drawback of this
method is clearly the large simpliWcation of protein shape.
While it may work Wne for single helices or highly symmet-
rical helix bundles [85,86], it is not suitable to deal with
membrane proteins of more irregular shaped cross sections.
The program code has been implemented in the GRO-
MACS package and recent applications of this method can
be found in [8789]. We consider the newer mdrun_hole
(see below) procedure superior.
Introduced in 2002 [90], mdrun_hole is a further devel-
opment of the cylinder approach. One still starts with a cyl-
inder as a Wrst approximation of the protein shape and
deletes all lipids with phosphorus inside that cylinder, but
then the Connolly surface of the protein itself is used as ref-
erence boundary to drive out lipids and water molecules
(see Fig. 2c), creating a tailored cavity to contain the pro-
tein [9198]. The program code is part of the GROMACS
package and additionally requires GRASP [99] or another
program to generate the solvent-accessible protein surface
in a format that can be read by GROMACS. The process
leads to systems that have excellent lipidprotein packing
and require minimal time to equilibrate compared to
coarser methods. Cylinder and mdrun_hole can be applied
to both water-free and fully hydrated systems.
In practice, a drawback of this procedure is that manual
intervention is often required: mdrun_hole has quite a few
input parameters that need to be speciWed but there is no
standard setting that works for every system. Instead
parameters like force constants for repulsive potential, sur-
face deWnition (atoms to consider, probe radius), oVsets
(deWning the eVective position of the surface boundary),
and water repulsion parameters may need to be Wne-tuned
for each system individually. Lipids can also become
trapped on their way out, in small cavities created by clus-
ters of side chain atoms, and must be removed manually. If
parameters for pressure and force constants are not chosen
properly (constant pressure and appropriately strong force
constants) and/or the number of lipids deleted initially is
too small, it is possible that some of the lipids may Xip over,
with their tails pointing towards the water phase. Con-
straints on lipid z coordinates can be used to prevent lipid
Xipping.
480 C. Kandt et al. / Methods 41 (2007) 475488

Fig. 2. As discussed in the text the preparation of a bilayer can be done by deleting lipids within the range of a simple protein lipid distance cut-oV (a), by
applying a repulsive potential to drive out lipid and water from the area to be occupied by the protein as in the cylinder approach (b), or with mdrun_hole
(c). Alternatively the membrane can be constructed around the protein lipid by lipid, taking structures from a pool of hydrated lipid conformations (d).
We introduce two approaches that involve scaling of the simulation system: one method starts by placing copies of lipids on a 2D grid and then scales
down the box size stepwise with energy minimization and short molecular dynamics steps in between (e). The other uses a pre-equilibrated bilayer, which
is expanded within the xy-plane. Lipids within a given cut-oV distance are removed and the system is brought back to natural dimension by a series of scal-
ing steps of the lipid xy positions and energy minimizations (f). (Figure created using RasMol, VMD and XaraX1).
 C. Kandt et al. / Methods 41 (2007) 475488
2.4.2.1.3. Build bilayer around protein. In contrast to the
methods discussed so far, this procedure does not use an
existing bilayer, but instead constructs the membrane
around the protein lipid by lipid (Fig. 2d). Originally intro-
duced in 1994 [20,100102], this approach starts by placing
large van der Waals spheres in two parallel layers around
the protein. These initial lipid spheres are suYciently sized
to contain the hydrated lipid head groups and are kept at
the average location of the lipid head groups, based on ref-
erence data from pure bilayers. Next, lipid molecules are
randomly chosen from pre-equilibrated lipid libraries and
placed with their head groups inside the initial van der
Waals spheres. Steric clashes are minimized using a rigid-
body conformational search technique, in the course of
which lipids are randomly rotated (around the Z axis) and
translated (in the xy-plane) within their initial spheres. The
method is implemented in the CHARMM package [36,103].
Even though lipid head groups are surrounded by a thin
layer of water molecules a later solvation step is required to
fully hydrate the system. Recent examples of this technique
can be found in [104110].
One could also start with a solution of lipids and the
protein and wait for the lipids to assemble around the pro-
teins. It has been shown that bilayers are formed on a time
scale of ca. 100 ns, currently expensive computationally but
within reach [111,112], and several papers have shown that
surfactant micelles can be assembled around membrane
proteins within a reasonable time [113115]. Although we
are not aware of published examples of this method with
phospholipids and membrane proteins, this is a potentially
simple method with minimal manual bias but a high
computational cost.
2.4.2.2. Two eYcient new approaches to create starting
structures. One of the cornerstones of any scientiWc
endeavor is reproducibility and transferability of an experi-
mental result independently from the person carrying out
that particular experiment. Unfortunately, especially in gen-
erating starting structures for membrane protein simula-
tions, this can be problematic as the inXuence the individual
user has on the set up process can be large. It would there-
fore be desirable to aim for an increasingly automated
approach to set up simulations that minimizes the individual
inXuence and not only maximizes reproducibility and trans-
ferability of obtained results but also opens a door to large
scale simulation approaches similar in spirit to those in
high-throughput genomics and proteomics. The two new
eYcient approaches to create a starting structure we intro-
duce here might provide a step towards that goal. The auto-
mated nature of the techniques allow one to vary the
composition of a system and run dozens of simulations with
minimal manual intervention. One could test the eVects of
putting a single protein into diVerent lipid environments, or
look at the properties of diVerent mutants or structural con-
formations of a protein in an identical lipid environment.
Both techniques are based on the strategy consisting of a
few steps: construct a bilayer with lipid-lipid spacing much
481
greater than seen in a real bilayer; insert the protein; and
then incrementally compress the bilayer around the protein
with alternating steps of compression and energy minimiza-
tion (Fig. 2e and f). Both approaches allow the construction
of simulation system with a mixed composition of lipids.
We propose two methods to generate the starting lattice
of lipids; the Wrst is to build a regular grid by replicating a
single lipid, the second is to blow up a pre-equilibrated
bilayer. We also discuss two methods to compress the
resulting bilayers, either by scaling the atomic coordinates
of the system and energy minimizing and performing short
MD steps, or by translating the lipids laterally and energy
minimizing each step. We describe these steps in the context
of two distinct methods, but they are interchangeableone
could use the compression method of the second approach
with the de novo bilayer generation of the Wrst approach,
and vice versa. A set of scripts to accomplish these tasks are
available on our website at http://moose.bio.ucalgary.ca. A
graphical summary of the second procedure is shown in
Supplementary material, movie 1.
2.4.2.2.1. Construct bilayer on grid and scale box size.
The Wrst leaXet is generated by placing copies of a single
lipid (in an approximately extended conformation) on a 2-
dimensional grid, using the GROMACS utility genconf. As
an option, each lipid can be randomly rotated around its Z
axis, a step which may or may not be useful (lipid orienta-
tions are somewhat correlated in a real bilayer [116]). When
preparing a bilayer for the insertion of a small protein, the
grid spacing can be chosen to allow the protein to Wt
between lipids, whereas for larger proteins a spacing of
510 times the estimated area per lipid of an equilibrated
bilayer is appropriate. To generate the second leaXet, the
starting leaXet is copied and then Xipped by rotating it 180
degrees around either the X or the Y axis (using editconf). It
is tempting to use a reXection of all atomic coordinates
through the plane of the bilayer, but this would invert the
stereochemistry of the phospholipids. The two leaXets are
translated to provide approximately the correct bilayer
thickness [117], then centered in a new box of appropriate
size. The X and Y dimensions of this box can be exactly cal-
culated from the lattice spacing used during generation of
the Wrst leaXet, whereas the Z dimension can take an arbi-
trary value (suYcient to encompass the whole protein).
The protein is copied into the box containing the two
leaXets, with the bilayer and protein positioned appropri-
ately along the Z axissee Section 2.4.1. Next, any lipids
that overlap with the protein are removed using a protein-
lipid interatomic distance cut-oV. This process can be auto-
mated, but care must be taken to ensure that a per-leaXet
distribution of lipids consistent with the shape of the
protein is maintained.
Next, bilayer and box size are compressed by a series of
scaling steps alternating with energy minimization. A simi-
lar technique was used previously to estimate the appropri-
ate area per lipid of a simulated bilayer and peptide system
[118]. The X and Y coordinates of the atoms, and the X and
Y box dimensions, are scaled (with editconf) using a scaling
482 C. Kandt et al. / Methods 41 (2007) 475488

factor less than one (0.95 is appropriate), which laterally
compresses the entire system. Subsequently energy minimi-
zation and/or a few hundred steps of molecular dynamics
are done with strong restraints on all protein atoms to
restore the proper geometry of all molecules. The corre-
sponding reference structure is generated at each cycle by
centering the starting protein structure in a box corre-
sponding to the box dimensions at the current cycle of com-
pression. The application of very strong position restraints
will then return the distorted protein to its original shape
while keeping it centered in the new box. This sounds dras-
tic, but it may be useful for avoiding lipid-protein overlap
during compression.
By comparing the area per lipid of the initial expanded
bilayer to the experimental area per lipid, an inXation fac-
tor of the expanded bilayer can be estimated and used to
calculate the number of compression cycles needed to
arrive at an optimum area per lipid. Alternatively, the area
per lipid can be estimated at each step (see below).
Due to the potentially long times (on the order of tens of
nanoseconds) required to allow the randomly oriented lip-
ids to properly equilibrate this technique may be of greatest
utility (compared to the second method, below) for auto-
mating the set up of small systems with relatively few lipids,
systems in which the bilayer is expected to be highly per-
turbed by the protein, or lipid types for which equilibrium
bilayer structures are not already available.
2.4.2.2.2. Use existing bilayer and scale lipid positions.
This is the second approach we introduce (Fig. 2f). The
starting point is a pre-equilibrated bilayer onto which
the protein has been superimposed so that the protein has
the desired orientation in the lipid bilayer but overlaps with
lipids. This system cannot be energy minimized due to the
extreme overlap. The bilayer is then expanded by translat-
ing lipid molecules and scaling the lateral dimensions of the
box size using a common scaling factor. Based on that scal-
ing factor a 2-dimensional translation vector is calculated
for each lipid. New lipid coordinates are calculated by
applying this translation vector to the old lipid coordinates
for each lipid (Z remains unchanged). The new X and Y
dimensions of the box are simply the old box dimensions
multiplied by the scaling factor.
The protein is centered in the new bilayer plane (based
on its center of mass) but remains otherwise unchanged.
Lipids within a deWned phosphorus-protein C distance
can be deleted. The number of lipids removed is reported
separately for the upper and lower leaXets.
Next a series of compression steps are performed
(using whole-lipid translations calculated as above, this
time with a scaling factor less than 1; 0.95 has been used
successfully so far), slowly bringing the system back to
more natural dimensions. After each scaling step the system
is energy-minimized to eliminate clashes. The protein is
constrained so its entire structure does not change.
To decide when a suYcient number of scaling and mini-
mization steps have been performed, the area per lipid is
calculated at each step, enabling comparison to the refer-
ence equilibrium value for a given lipid species (see Fig. 3).
In order to determine the area per lipid, the area occupied
by the protein must be calculated Wrst. To do this, a rectan-
gular grid is placed on the protein in the xy-plane and all
protein atoms in between two speciWed planes are taken
into account. These planes are deWned by the average z
positions of lipid phosphorus atoms in the upper and lower
leaXet. If a grid cell contains any protein atoms it is consid-
ered occupied. The area of these grid cells is summed to give
an approximation of the area occupied by the protein. As
van der Waals radii of the atoms are not considered in the
calculation, we found a grid size of 5 gave the best results.
The area per lipid for each shrinking step is combined in a
simple plot. When compared to the equilibrium value of a
pure bilayer the choice of the appropriate structure is
straightforward. Our approximation of the protein area
generally tends to underestimate the real area per protein;
therefore we suggest using the lipid structure from the
shrinking step immediately prior to the point where the
area per lipid is closest to the reference value. For
membrane proteins with a more irregular shape of the

Fig. 3. One way to determine the number of necessary compression steps is to monitor the area per lipid. To that end the area per protein is computed Wrst:
only protein atoms in the range between the upper and lower leaXets average phosphorus positions (white) are considered (left), in a 2D grid approach to
estimate the area per protein (middle). Area per lipid can then be calculated and compared to the reference value for a given lipid species (horizontal line
at 0.53 nm2). The recommended structure to use is marked by a grey circle (right). (Figure created using VMD, XMGRACE and XaraX1).
 C. Kandt et al. / Methods 41 (2007) 475488
transmembrane region the approximated area per lipid can
be calculated separately for upper and lower leaXet. Figs. 2f
and 3 illustrate the actual application of this method to the
ABC transporter system BtuCD-F. The whole procedure of
inserting the protein into the bilayer was completed after
less than one hour.
2.4.3. Solvate system
In the previous section, a membrane protein was embed-
ded in a bilayer but in most cases water was not present.
Because water in the water and lipid headgroup region gen-
erally equilibrates fast, on a 100 ps time scale, water can be
added relatively easily. The key issues are how to deal with
water in the hydrophobic interior of the membrane and
how to solvate cavities in proteins that do not easily
exchange water molecules with the bulk solution. Solvation
techniques use a geometrical rather than a thermodynami-
cal approach to add water. As the partitioning of water into
a bilayer is unfavorable enough to see any water inside the
hydrophobic part of a bilayer, the goal is therefore a suY-
ciently solvated starting structure with no water molecules
in the hydrophobic section. Even though water will diVuse
out of this section given enough equilibration time it is
more practicable and computationally more eYcient to
remove these water molecules from the starting structure.
Supplementary movie 2 illustrates the slow but spontane-
ous process of draining water from the center of the bilayer
if it is not removed manually (2.5 ns, in this example). Any
cavities in the bilayer suitable to contain water but still
empty after the solvation step will Wll up during the simula-
tion.
2.4.3.1. Delete redundant waters based on distance cut-oV.
In this approach the system has already been solvated
and water has penetrated the hydrophobic interior of the
membrane. Distance cut-oVs between diVerent subsets of
the simulation system are then applied to identify mis-
placed water molecules using standard molecular graphic
software like e.g., RasMol or VMD.
Misplaced waters are found in proximity of the lipid
tails. However, simply selecting water nearby lipid tails will
not suYce as this would also aVect waters around lipid
head groups or inside the protein. Further subsets need to
Fig. 4. To avoid misplaced waters in the hydrophobic section of the bilayer an eYcient way of solvating the system is to increase the van der Waals radii of
some lipid tail atoms (a). That way any cavities in this region of the bilayer are eliminated (b). No water molecules are placed here but at the same time
both lipid head groups (grey) and protein interior are suYciently hydrated (c). (Figure created using MolMol and XaraX1).
483
be deWned protecting these water molecules from being
removed. A possible way to do this would be deWning a
suitable number of nearby subsets allowing to identify
only water molecules close to the lipid tails but at the same
time not adjacent to lipid heads or the protein interior.
With this technique it is possible to delete the major part
of misplaced water molecules but the distance cut-oVs used
must be chosen carefully to ensure lipid head groups
remain suYciently solvated and no cavities are formed
towards the water phase. Experimenting with diVerent set-
tings is a good idea. Remaining waters have to be deleted
manually.
2.4.3.2. Delete redundant waters based on their z position.
Like in the Wrst approach the system has already been
solvated and misplaced waters are present. The z range of
the hydrophobic part of the bilayer is determined and all
water molecules inside this range of z coordinates are
deleted. The disadvantage is obvious: protein-internal
waters will also be removed which can severely aVect the
proteins structural stability. One idea to circumvent this
problem is to delete waters within said z range Wrst and sub-
sequently re-solvate the protein interior using cavity detec-
tion and hydration tools such as VOIDOO [119] which is
available at the Uppsala software factory (http://
alpha2.bmc.uu.se/~gerard/manuals/).
2.4.3.3. Avoid putting waters in wrong places. Programs that
solvate a simulation system usually perform a relatively
simple form of cavity analysis to decide where to place
solvent molecules. In GROMACS, the data for the van
der Waals radii of the diVerent atoms are stored in a sepa-
rate ASCII Wle vdwradii.dat which is read by the solvating
tool genbox. These radii can be modiWed to change the
behavior of genbox. As illustrated in Fig. 4, the idea is to
increase the van der Waals radii of some of the lipid tail
atoms (e.g., 4 for the last 4 carbon atoms of each of the
two lipid tail) to eliminate any cavity in the hydrophobic
section of the bilayer that otherwise could be Wlled with
water. This is done for the solvation step only (Fig. 4b).
This produces a fully hydrated system where both
lipid head groups and protein interior are suYciently sol-
vated but no water molecules have been placed in the
484 C. Kandt et al. / Methods 41 (2007) 475488

hydrophobic region of the lipid tails (Fig. 4c). No further
working steps are required.
2.5. Simulation parameters
Among the numerous run parameters the most crucial
ones for membrane protein simulations include the type of
pressure coupling, the way electrostatics are treated and the
actual length of the simulation.
2.5.1. Pressure coupling
There are signiWcant theoretical issues involved in pres-
sure coupling in interface systems [120]. A detailed compar-
ison of several algorithms, some more rigorous than others,
for pressure coupling as well as some pragmatic approaches
showed no substantial diVerence in tests of pure lipids [68],
and we take a practical approach here for the sake of sim-
plicity.
Roughly, there are three diVerent methods of pressure
coupling: Isotropic, semi-isotropic and anisotropic (see
Fig. 5). With isotropic pressure coupling the single contri-
butions in x, y, and z direction are coupled, thus only a pro-
portional scaling of the system is possible (Fig. 5a).
Generally this leads to very small changes in box size due to
the incompressibility of water. These changes are often neg-
ligible. When larger changes occur, it usually indicates an
insuYcient solvation of the system or mistakes during mod-
eling. In regard to membrane protein simulations it is
important to keep in mind that isotropic pressure coupling
does not allow Xuctuations in the surface area, which is a
key feature of lipid bilayers, and does not specify a surface
tension. As such it is inappropriate for membrane simula-
tions. One possible method to deal with this is to impose a
constant surface area, calculated from the area per lipid
value for the particular lipid species of interest, or a con-
stant volume. Unfortunately knowledge of the right area
per lipid is very hard to obtain and actually not possible to
get for arbitrary systems. Furthermore, this approach will
not allow reproducing eVects such as temperature depen-
dent swelling of the bilayer.
Semi-isotropic pressure coupling does allow area Xuctu-
ations. In this case, only the pressure contributions in x and
y direction are (isotropically) coupled, but the one in z is
not (Fig. 5b). It is important to stress here that having the x
and y pressure contributions coupled does not imply a con-
stant surface area: if the size of the simulation box needs to
be adjusted this will aVect both dimensions equally to allow
area Xuctuations without changing the aspect-ratio of the
box. Semi-isotropic is the recommended type of pressure
coupling for membrane protein simulations. With the
united-atom parameters in GROMACS a reference pres-
sure of 1 bar in both xy and z is appropriate, and corre-
sponds to a realistic pressure [121]. Simulations using
CHARMM parameters sometimes use diVerent values in
xy to impose a non-zero surface tension. This corresponds
to a membrane under tension but is related to force Weld
issues that are still under investigation.
Although anisotropic coupling also enables Xuctuations
of the membrane area it should be used carefully: as there is
no coupling between any of the directions of pressure con-
tributions, this can result in large deformations of the
whole simulation system (Fig. 5c).

Fig. 5. There are three types of pressure coupling: isotropic pressure coupling has all three pressure contributions (in x, y, and z, assuming the box has to
remain rectangular) coupled, permitting only a proportional scaling of the simulation system. Only small Xuctuations can occur this way, Xuctuations in
surface area are not possible (a). Semi-isotropic pressure coupling does allow area Xuctuations as here only the pressure contributions in x and y direction
are coupled together but the one in z is not (b). Anisotropic pressure coupling too enables Xuctuations of the membrane area but can also lead to large
deformations of the simulation system as there is no coupling between any of the directions of pressure contributions (c). (Figure created using XaraX1).
 C. Kandt et al. / Methods 41 (2007) 475488
2.5.2. Electrostatics
A key issue in molecular dynamics simulations is the
treatment of electrostatic interactions. This especially con-
cerns lipid bilayer systems, as phospholipids have a very
high interfacial charge density and regions of low dielectric
constant where electrostatic interactions are largely
unshielded. In biomolecular simulations, there are three
main ways to compute electrostatic interactions: cut-oV
based techniques, lattice-sum techniques of which particle
mesh Ewald (PME) is by far the most popular [122,123],
and reaction Weld approaches (RF) [124]. The issue is to cal-
culate energies that decay as 1/distance, a common problem
in physics, and other methods that have not found routine
use in MD simulations yet exist [125].
Cut-oV approaches neglect long-range Coulombic eVects
by truncating the electrostatic forces at a speciWc cut-oV dis-
tance, which is typically between 1.0 and 2.0 nm, potentially
giving considerable savings in computational cost. However,
such approximations are rather drastic and have a signiWcant
eVect on the systems properties [68,126]. As an improvement
over straight cut-oV methods, shifting functions can be
applied where artiWcial correlation eVects are partly removed
by smoothing the interaction energy to zero either within the
full cut-oV range or over a limited region [68].
The basis of PME is an interpolation of the reciprocal-
space Ewald sum. The simulation box is replicated to all
sides, and all of the interactions in this periodically repli-
cated system are summed. The eVect of long-range electro-
static interactions is included. A potential concern with this
method is that due to its periodicity, PME may induce an
artiWcial ordering that enhances the systems stability.
Detailed tests of what should be an extreme casea system
consisting of an alemethicin helix bundle with six parallel
helices, all aligned with their helical dipoles in the same
directiondid not show a strong eVect, however [127]. In
principle, it is possible to modify a lattice sum to be peri-
odic only in the plane of the membrane, a natural symmetry
for membranes. This has been tested for the same alamethi-
cin bundle [128], but the main results are inconsistent with
earlier results. This may be due to the somewhat complex
corrections required. Nevertheless, this may be a promising
approach with some further development and testing.
Reaction Weld approaches are another technique that
considers the eVect of long-range electrostatic interactions.
They add a correction term to the cut-oV result based on a
continuum electrostatics description of the solvent outside
the cut-oV sphere. This method was originally developed for
the use in homogenous systems such as liquid simulations.
As recently pointed out by Anezo and coworkers, none
of these methods are perfect for the simulation of interfa-
cial systems: cut-oV methods induce artiWcial orderings;
PME enhances periodicity; while RF approaches ignore the
heterogeneous nature of the membrane [68]. However,
against this background it seems that today PME would be
the recommended choice bearing the least drawbacks. The
choice is not arbitrary; a particular force Weld really should
be developed for use with one of these methods, as the
485
accuracy of the force Weld is not maintained when switching
between diVerent methods to calculate electrostatic interac-
tions. An interesting future avenue is to extend the idea of a
reaction Weld by combining a cut-oV with a solution of an
approximate description of the system as spatially varying
dielectric constants. This idea is not new and was in fact
used in one of the Wrst simulations of a lipid bilayer [129],
but seems worth revisiting for more complex systems.
2.6. Equilibration length
The simulation time required to equilibrate a membrane
protein simulation system depends on many factors. Two
major ones are the actual system size (the bigger the system,
the slower equilibration) and the starting structure used.
For example, how big is the degree of disturbance caused
by the insertion of the protein? How good is the quality of
the protein model used? Is the system solvated suYciently?
Regarding the protein, this concerns both external and
internal hydration.
It is generally useful to keep in mind that 1020 ns are
required to equilibrate pure DPPC phospholipid bilayers
[68], consistent with our experience with other lipids if the
starting structure is reasonably close to the equilibrium struc-
ture [130]. Major rate limiting steps are the rotational and
lateral diVusion of the lipids, thus the system set up should be
reWned to a point where this is minimized at all costs. A time
scale of at least nanoseconds is a good practical starting time
for a typical membrane equilibration run, but will often be
too short. Protein coordinates should be kept restrained at
this stage. Progress in equilibration can be judged by moni-
toring the dimensions of the simulation box, lipid properties,
and energies. The total potential energy often becomes stable
much faster than anything else and is not a reliable indicator
of equilibration, but component energies such as lipidlipid
interaction energy, lipidprotein, lipidwater, and protein
water can be more informative. Generally large scale defor-
mations in the bilayer must be avoided. Such deformations
may be obvious holes, but more typical they could be
strongly bent bilayers or lipids that have left the bilayer (a
very rare event in equilibrium). If they occur nevertheless the
distribution of lipids in the two leaXets should be checked for
asymmetries: removing too many lipids from one leaXet is a
common source of error in membrane protein simulations.
This leads to strong deformations of the bilayer which are
often misinterpreted as an eVect of the membrane protein,
which they clearly are not. Unfortunately it is diYcult to
suggest exact criteria, but one of the most common problems
in the literature on membrane protein simulations is lack of
equilibration and invalid conclusions drawn from such
simulations.
2.7. Analysis
ReXecting the large variety of possible membrane protein/
peptide systems and speciWc scientiWc questions accompany-
ing each of them, there is no common way how to analyze
486 C. Kandt et al. / Methods 41 (2007) 475488
such simulations in general. In one case one might focus on
dynamics of certain micro-environments inside the protein
[78,83] while in another case large scale rearrangements of
whole protein subunits are more important [94]. Apart from
checking fundamental properties such as stability of the
bilayer and making sure known experimental data is repro-
duced satisfyingly well each system will present unique chal-
lenges in choosing and/or creating appropriate analysis tools.
Although this is outside of the scope of this review, the pal-
ette of membrane protein simulation data comparable to
experimental observables is broad [70,131,132]. For the pro-
tein part it ranges from direct comparison of structural data
determined by X-ray crystallography, NMR and electron
microscopy over distance data (residue residue distance as
determinable by double spin-labled EPR or distance matrices
measured by NMR), average orientation of helices (linear
dichroism), secondary structure content (circular dichroism)
and radius of gyration (SAXS) up to predicting the eVect of
certain residue mutations, be it the identiWcation of possible
knock out mutants or predicting the spectral eVects of cer-
tain residue replacements [78]. For the bilayer part however,
the lack of accurate high-resolution experimental data still
restricts comparable observables to parameters such as 1D
density proWles or area per lipid [50].
3. Concluding remarks
In this paper we have reviewed practical aspects of mem-
brane proteins simulations, with a particular emphasis on
diVerent approaches to set up such simulations. We intro-
duced two new methods to generate a starting structure.
These methods may make the process more eYcient and
eventually allow large scale simulations of either many
diVerent models or many diVerent proteins from the data-
base in an unbiased and automated way. In principle it
should not matter which method is used to prepare a mem-
brane protein simulation, as long as the system has been
equilibrated long enough and known experimental data are
reproduced suYciently well, but in practice there are sub-
stantial diVerences. We also presented a number of ques-
tions and issues that should be considered prior to any
modeling or simulation.
It is important to keep in mind what the worst elements
of a simulation arethere is no point in optimizing a tech-
nical detail of a particular algorithm or parameter if sam-
pling in the simulation is inadequate or a starting structure
of a protein is an approximate homology model. Ulti-
mately, the goal of most simulations is to provide useful
insights into the problem of interest. Only if such a simula-
tion both meets technical standards and does provide new
insights is it useful.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.ymeth.
2006.08.006.
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