An ALCHEMBED tutorial
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common-files
LICENSE.md
README.md
try-alchembed.sh

README.md

An ALCHEMBED tutorial.

Release notes

One can obtain this tutorial from two places.

  1. As a tarball as part of the Supplementary Information of the ALCHEMBED paper. This version is therefore easier to find and will be maintained indefinitely, but will not evolve with time. The paper and supporting information is availaible from here: doi:10.1021/ct501111d
  2. As an open GitHub repository. Changes will be made to this repository with time to reflect e.g. changes in GROMACS functionality.

To obtain the repository, assuming you have git installed, issue.

git clone https://github.com/philipwfowler/alchembed-tutorial.git 

which will create a directory called alchembed-tutorial and download the files.

Objective

To demonstrate how the ALCHEMBED method can embed different membrane proteins into lipid bilayers.

Citing

This tutorial accompanies the following paper

@article{Jefferys2015,
author = {Jefferys, Elizabeth and Sands, Zara A and Shi, Jiye and Sansom, Mark S P and Fowler, W},
title = {{Alchembed : A computational method for incorporating multiple proteins into complex lipid geometries .}},
journal = {J Chem Theo Comp},
volume = {11},
pages = {2743–2754},
doi = {10.1021/ct501111d},
year = {2015}
}

and allows the user to embed the five different membrane proteins shown in Figure 3 into a simple lipid bilayer comprising 512 POPC lipids. As in the paper, each protein can be embedded using either a fully-atomistic forcefield (CHARMM27) or a coarse-grained forcefield (MARTINI2.2).

If you use this tutorial please cite the above paper in your work.

Pre-requisites

As in the paper, all the simulations are performed in GROMACS, although in principle the method should work equally well in AMBER, NAMD or CHARMM since all these codes include van der Waals soft-core functionality.

Instructions

All the GROMACS commands have been stored in a bash script, try-alchembed.sh, that is in the root of this repository. All of the files required for the simulations are stored in the common-files/ directory. The five test proteins are referred to using these slightly shortened names ['nbar','pla2','cox1','kcsa','ompf'] and the forcefield is specified as one of ['at','cg'].

Take for example the 'nbar' protein in a 'cg' representation.

ls common-files/nbar-cg.*
common-files/nbar-cg.itp common-files/nbar-cg.pdb
common-files/nbar-cg.ndx common-files/nbar-cg.top

There are four files in common-files/ specific to this protein/forcefield combination. (The atomistic simulations have a fifth file that foo-at_posre.itp that specifies which protein atoms to position restrain during the simulation). The PDB file contains the intial coordinates of the lipids, protein and water. Please visualise this using VMD/PyMol/Chimera to satisfy yourself that many of the protein and lipid beads clash. To provide a more stringent test you could also move the protein relative to the bilayer, rather than use the conformation provided. The NDX file is contains the index groups; these are referred to in the MDP file (see below). Finally the TOP file specifies the composition of the system and the location of the ITP files (including the protein one listed above) which describe the connectivity of the different molecules.

The ALCHEMBED process has two steps; the first is a short energy minimisation. The run parameters for this are specified in

less common-files/em-cg.mdp

The second step is a short 1000 step GROMACS MD simulation where the van der Waals interaction between the protein and the rest of the system is described by a soft-core van der Waals potential. The run parameters for this are here

less common-files/alchembed-cg.mdp

The strength of the (soft-core) van der Waals interaction between the protein and the rest of the system is described by a coupling parameter, lambda. Initially, lambda is zero and there are no forces between the protein and the rest of the system. Here lambda increases by 0.001 for 1000 steps, thereby smoothly "turning on" the interactions between the protein and the rest of the system. During this process the position of the protein beads (or atoms) are restrained and as lambda increases the lipid beads (or atoms) move out of the space occupied by the protein.

The try-alchembed.sh script takes two arguments (the script is commented also if you'd like to look inside). The name of the protein (taken from the list above) and the forcefield. Hence to run it type

./try-alchembed.sh nbar cg

and assuming you have GROMACS in your $PATH etc, then it should perform the short energy minimisation and then the embedding simulation. On a single core of at Intel Xeon E5 processor (c. 2014) this took 13 seconds. The larger proteins and the atomistic cases will take longer (nbar at took around 15 min on the same processor).

All the regular GROMACS output files are stored in protein/forcefield/, i.e. nbar/cg/ in this case (the script automatically creates the directory if it doesn't exist). The files ending in -em are from the energy minimisation run and those ending in -alchembed are from the embedding run. Hence to examine the result of the above short run,

cd nbar/cg/
ls
 nbar-cg-alchembed.cpt      nbar-cg-alchembed.trr      nbar-cg-em.log
 nbar-cg-alchembed.edr      nbar-cg-alchembed.xtc      nbar-cg-em.mdp
 nbar-cg-alchembed.gro      nbar-cg-alchembed.xvg      nbar-cg-em.tpr
 nbar-cg-alchembed.log      nbar-cg-alchembed_prev.cpt nbar-cg-em.trr
 nbar-cg-alchembed.mdp      nbar-cg-em.edr
 nbar-cg-alchembed.tpr      nbar-cg-em.gro
vmd -pdb ../../common-files/nbar-cg.pdb -xtc nbar-cg-alchembed.xtc

assuming you have VMD installed and in your $PATH. A good way in VMD of seeing what is going on is to first create a Graphical Representation of the protein. A Transparent QuickSurf works well. Then create a Graphical Representation that displays the number of lipid/water beads/atoms within sigma of the protein. Opaque VDW is good here. For AT simulations the Selected Atoms would be

not protein and within 2.4 of protein

and then check "Update Selection Every Frame" under the "Trajectory" tab. For CG a difficulty is that the "protein" keyword does not work. Instead try

resname W POPC and within 4.7 of (not resname W POPC)

Also, you will need to remove the jumps across the periodic boundary conditions in any AT sim via trjconv, which is part of the GROMACS packages so should be in your $PATH. For example, for nbar at

trjconv -f nbar-at-alchembed.xtc -s nbar-at-alchembed.tpr -pbc mol -o nbar-at-alchembed-nojump.xtc

then load this XTC file into VMD instead

vmd -pdb ../../common-files/nbar-at.pdb -xtc nbar-at-alchembed-nojump.xtc 

Extensions

To further convince yourself that the ALCHEMBED process has successfully embedded the test proteins into the POPC bilayer, you could use the final GRO file (e.g. nbar-cg-alchembed.gro) to start a standard MD NpT simulation.