1. Bales Of Amber Mac Os 11
2. Bales Of Amber Mac Os X

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1. References
   

Lipidsare a crucial component of lipid bilayers and play an important role inmany cell signaling and physiological processes. Condensed phasemolecular dynamics software packages like Amber are now able tosimulate a variety of biomolecules, including lipids.

This Tutorial

In this tutorial, we present a step-by-step guide to setting up a lipidbilayer system and running molecular dynamics with Amber and theLipid14 force field. We assume that you are using a Unix system withAmber14 successfully installed. We also assume you have run basic Ambermolecular dynamics simulations before.
This tutorial shows how to simulate a lipid bilayer and a lipid bilayerwith a membrane-bound protein. It is often important to understand thedynamics of the bilayer itself before proceeding with protein systems.This tutorial goes on to provide details of how to set up membranebound proteins systems using Amber and Lipid14.
Warning
This tutorial describesrunning lipid molecular dynamics simulations for instructionalpurposes. The settings in this tutorial are designed for this tutorialand will need to be adapted to other systems. A detailed understandingof each step will help to make informed decisions for other systems.

Prerequisites

1. Amber and AmberTools version 14.0 installed on a Unix based machine (Linux of Mac OS).
   
1. Lipid14 force field
   Lipid14 is included in AmberTools 14 by default.
2. charmmlipid2amber.py
   This is included in AmberTools 14 update 1.
2. VMD 1.9.1
   A molecular visualization program.

Please cite your use of the Lipid14 force field with:
Dickson, C.J., Madej, B.D., Skjevik,A.A., Betz, R.M., Teigen, K., Gould, I.R., Walker, R.C., 'Lipid14: TheAmber Lipid Force Field', J. Chem. Theory Comput., 2014, 10(2), 865-879.
DOI: 10.1021/ct4010307
Amber 14 includes Lipid14 [1], a modular lipid force for tensionless lipidphospholipid simulations. Lipid14 includes the modular chargederivation framework developed in Lipid11 [2] as well as areparameterization of key van der Waals and dihedral angles asperformed in GAFFlipid [3]. Lipid14 has been extensively tested andvalidated on six key lipid bilayer types. Lipid14's parameterizationstrategy is consistent and compatible with the approach taken by otherpairwise-additive Amber force fields. Therefore, Lipid14 is, in principle, fullycompatible with the other biomolecular force fields included in Amber.
The currently supported lipid 'residues' are listed below as well asthe validated lipid bilayers. For reference, we also list the previous lipids in GAFFlipid and Lipid14. Additional head groups and tail groups as well as other lipid bilayer components such as cholesterol will be included in upcoming updates of the Lipid14 force field.

	Description	LIPID14 Residue Name
Acyl Chain	Lauroyl (12:0) Myristoyl (14:0) Palmitoyl (16:0) Stearoyl (18:0) Oleoyl (18:1 n-9)	LA MY PA ST OL
Head Group	Phosphatidylcholine Phosphatidylethanolamine	PC PE

	Description	LIPID11 Residue Name
Acyl Chain	Palmitoyl (16:0) Stearoyl (18:0) Oleoyl (18:1 n-9) Linoleoyl (18:2 n-6) Linolenoyl (18:3 n-3) Arachidonoyl (20:4 n-6) Docosahexanoyl (22:6 n-3))	PA ST OL LEO LEN AR DHA
Head Group	Phosphatidylcholine Phosphatidylethanolamine Phosphatidylserine Phosphatidic acid (PHO4 -) Phosphatidic acid (PO4 2-) R-phosphatidylglycerol S-phosphatidylglycerol	PC PE PS PH- P2- PGR PGS
Other	Phosphatidylinositol Cholesterol	PI CHL

Name	GAFFlipid Molecule Name
1,2-dilauroyl-sn-glycero-3-phosphocholine 1,2-dimyristoyl-sn-glycero-3-phosphocholine 1,2-dioleoyl-sn-glycero-3-phosphocholine 1,2-dipalmitoyl-sn-glycero-3-phosphocholine 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine	DLPC DMPC DOPC DPPC POPC POPE

In order to simulate a lipid bilayer, it is necessary to first obtain astarting structure in PDB format that can be processed by Amber's LEaPprogram. There are several accessible options for building a lipidstructure:

• CHARMM-GUI Lipid Builder
  A simple, internet based solution to generating lipid bilayer structures as well as membrane-bound protein structures
  
• VMD Membrane Builder plugin
  A plugin is available for the Visual Molecular Dynamics (VMD) programthat can currently build POPC and POPE bilayer systems. A tutorialexplaining how to build lipid bilayers as well as insert membrane-boundproteins is available on the VMD web site.

In this tutorial, you will generate a lipid bilayer structureusing the CHARMM-GUI web site, and then convert that PDB formatted fileto a file formatted for Amber's preparatory program LEaP. Forsimplicity, you will use the web based CHARMM-GUI to build an initiallipid bilayer structure. For this tutorial, you will build a simpleDOPC lipid bilayer with 128 lipids and use the Lipid14 force field forproduction MD.

Go to the CHARMM-GUI Lipid Builder
Membrane Builder
Choose 'Membrane Only System'.
System Size Determination
Choose a 'Heterogeneous Lipid' system.
Your system will be rectangular and periodic in the X, Y, and Zdimensions. You will be simulating a patch of bilayer with periodicboundary conditions.
Choose a 'Rectangular' box type.
Choose an appropriate hydration number for your lipid type to definethe box Z length. This is important for a properly solvated bilayersystem. It is suggested that you review the literature for appropriatehydration numbers for various lipids.
For DOPC, use '37' waters per lipid, in excess of the experimental 32.8 waters per lipid [4].
Select 'Numbers of lipidcomponents' for your system to define the box XY length. This willcreate a system based on the surface area per lipid.
Now enter the '# of Lipid on Upperleaflet' and '# of Lipid on Lowerleaflet'. See the CHARMM-GUI table below for a list of currently supported CHARMM-GUI lipids.
For DOPC, set '64' on the lower leaflet and '64' on the upper leaflet.
Click 'Show the system info' and verify the system properties are correct.
Continue to the next step.
Component Building Options
Depending on your lipid type and interfacial environment, you will wantto include an ion concentration. It is important to include ions due tothe inherent charge of the lipid head groups and their interaction withwater solvent. Some systems may have special ionic concentrationrequirements: for example in protein ion channel systems.
For DOPC, set the ion concentration at '0.15' M 'KCl'. Choose the 'Monte-Carlo' ion placing method. Continue to the next step.
Building Ion and Waterbox
The next two steps build the lipid bilayer system into a single structure file.
Continue to the next step.
Assemble Generated Components
Continue to the next step.
Determine System Size and Equilibration Options
At this point, the generated lipid, waters, and ions have been combined into a single structure PDB file.
Download the .tgz file and save it to your computer.
step5_assembly.pdb
From the charmm-gui.tgz file, the only file needed is the assembled PDBfile step5_assembly.pdb. This file should have the lipid bilayer, thewater molecules, and included ions. It is important to verify allchosen components are present.
Extract step5_assembly.pdb and save to your working directory.

Bales Of Amber Mac Os 11

Currently Supported CHARMM-GUI Lipids with Lipid14

CHARMM-GUI Lipid	Lipid14 Residue 1	Lipid14 Residue 2	Lipid14 Residue 3
DLPC DMPC DPPC DOPC POPC*	LA MY PA OL PA	PC PC PC PC PC	LA MY PA OL OL
DLPE DMPE DPPE DOPE POPE*	LA MY PA OL PA	PE PE PE PE PE	LA MY PA OL OL

*Can be built with VMD builder and processed with charmmlipid2amber.py
Now you have an initial lipid bilayer PDB file. However, there is no standard for structuring lipid PDB files.
Briefly, the Lipid14 format is as follows: The lipid is split intothree 'residues': two tail groups and one head group. Each residue inLipid14 has a specific residue name, atom names, and atom types. In aLipid14 PDB file, a phospholipid molecule is listed as a chain of threelipid residues in specific order: sn-1 tail group, head, sn-2 tailgroup. Each molecule consisting of a chain of three residues must beended with a TER card.
Lipid14 PDB File Format
The CHARMM-GUI PDB formatted file must be reformatted to file that follows the Lipid14 format that LEaP expects.

charmmlipid2amber.py

charmmlipid2amber.py
charmmlipid2amber.py is a robust residue and atom renaming andreordering script available with AmberTools 14 update 1. It is usedhere to convert the CHARMM-GUI PDB format structures into a PDB formatthat can be read with LEaP and Lipid14. If you do not have AmberTools14 update 1 available, you can download the zip file from this website. This can be placed in your Amber directory and unzipped there.
Warning
charmmlipid2amber.py has replaced the charmmlipid2amber.x script. It is included in AmberTools 14 update 1.
Now, to convert the CHARMM-GUI formatted PDB file to the Lipid14 formatted PDB file, use the script charmmlipid2amber.py.
charmmlipid2amber.py usage:
input_structure.pdb is the CHARMM-GUI formatted PDB file andoutput_structure.pdb is the Lipid14 formatted PDB file to be loadedwith LEaP.
For the 128 lipid DOPC system, convert the PDB file:
DOPC_128.pdb
After the script completes processing the PDB file, you should have a file DOPC_128.pdb ready to be loaded into LEaP.

Estimate Periodic Box Size

Due to the nature in which CHARMM-GUI builds lipid bilayers, there willbe lipids extending beyond the solvation layers in the X and Ydimensions. It is possible to better estimate the periodic boxdimensions by using the water molecules' coordinates for thismeasurement. This can be manually measured using a PDB structure viewerlike VMD.
However, we provide a simple bash/VMD script here to measure the periodic box dimensions here:
vmd_box_dims.sh usage:
Now calculate your box dimensions from the water molecules:
In this section, you will load the file named DOPC_128.pdb into LEaP todefine the topology and parameters for the lipid residues making upeach phospholipid. Use this method to prepare an Amber parametertopology file and initial coordinate file.
Start xLEaP:
In the LEaP command line, source the force fields you want to use.Lipid14 is designed to be compatible with the other pairwise-additiveAmber force fields.
Load the force fields:
With the processed file, DOPC_128.pdb, LEaP will load the structure, split the lipids into three units, and assign atom types.
Load the lipid structure file into a unit:
Note
There will be warningsabout lipids being split into different residues. This is normal LEaPbehavior. The residue sequence number may not correspond to theresidues in your PDB file.
Now we use the periodic box size as measured from the water molecules in our structure using VMD.
Add the periodic box to the system:
Save the Amber prmtop parameter and topology file and the inpcrd initial coordinate file for the molecular dynamics simulation:
Note
Pay close attention to any errormessages that LEaP provides when saving the parameter and topologyfile. Verify that there are no missing parameters.
DOPC_128.prmtop
DOPC_128.inpcrd
At this point the necessary files have been generated to run the actual simulation.
Quit LEaP and verify that the prmtop and inpcrd files were created.
Warning
Molecular dynamicssimulations of lipid bilayers is not trivial. It is suggested that youfirst review the literature regarding lipid bilayer simulations beforeproceeding. The method presented below is similar to that used in theLipid14 publication.
For molecular dynamics of lipid bilayer systems the following protocol can be used:

1. Minimization
2. Heating, holding the lipids fixed
3. Heating 2, holding the lipids fixed
4. 10X Hold, to equilibrate periodic box dimensions
5. Production with constant pressure

Minimization

The first step is essential to minimize and relax the initiallygenerated structure. This method of minimization is fairly common inAmber molecular dynamics simulations.
Note
The input files below are commented to provide an explanation of the various options used
01_Min.in
In order to run the minimization with pmemd, use the following command:
After the minimization is complete, you can read the mdout file for thedetails of the minimization. The important file for the next stage ofsimulation is the 01_Min.rst restart file, which will be used for theheating portion of the molecular dynamics.

Heating

After the initial minimization, slowly heat the system to productiontemperature. Choosing a production temperature is an important choicein the lipid bilayer simulation. Lipid bilayers haveexperimentally-measured phase transition temperatures from highlyordered gel-like phases to liquid phases. One major problem in lipidbilayer simulations is accurately simulating the phase transition oflipids.
For DOPC, a production temperature of 303 K is acceptable because it iswell above the phase transition temperature. The system was heatedthrough two sequential runs to 303 K while keeping the lipid fixed.First the system is heated to 100 K and then slowly to the productiontemperature. Use the following input for the first heating step:
02_Heat.in
Note that the Langevin thermostat is used for the initial heating. Inaddition, the nmropt=1 allows one to vary the system temperaturethroughout the heating. The lipid molecules are restrained using aharmonic restraint and the GROUP input section at the end of the inputfile.
To run the first heating moleculary dynamics, use the following command:
Unfortunately because of the size of the trajectory files, this tutorial web site cannot host these files.

Heating 2

The second phase of heating slowly increases the temperature to thedesired production temperature. The positions and velocities are readfrom the previous restart file. This time an anisotropic Berendsenweak-coupling barostat is used to also equilibrate the pressure inaddition to the use of the Langevin thermostat to equilibrate thetemperature.
03_Heat2.in
To run the second heating molecular dynamics, use the following command:

Hold

In order to equilibrate the system's periodic boundary conditiondimensions, it is necessary if you are using the GPU code pmemd.cuda, to run 5ns MD with a barostat. The system'sdimensions and density must equilibrate beforeproceeding with production MD. Because the periodic boundary condition boxdimensions are changing, it is necessary to increase the 'skinnb' valueand to restart the MD simulation after 500ns. This should avoid most'skinnb' errors.
The reason for this is that, for performance reasons, the GPU code does not recalculate the non-bond list cells during a simulation. If these cells change size, due to the box changing size, by too much then it will cause the code to halt with an error related to skinnb. Once the system is equibrated the box size fluctuations should be small and so this should not be an issue during production.
04_Hold.in
We run this hold production 10 times (5ns) to equilibrate the system before production MD. This can be scripted easily.
To run production of the lipid bilayer, use the following command and run the following:

Production

Actual production molecular dynamics can be run with the input fileshown below. Temperature is controlled here using the Langevin thermostatwhile pressure is controlled using the anisotropic Berendsen barostat.
Use this input file for the production of lipid bilayers:
05_Prod.in
To run production of the lipid bilayer, use the following command:

Using the restart files and this same input file this simulation can becontinued until the structure has converged. In particular, it iscommon to continue production until the bilayer and the structure'sarea per lipid has converged.
After production, there are several lipid bilayer parameters worthexamining including area per lipid, electron density profiles,deuterium order parameters, as well as others. Current lipid moleculardynamics literature present many methods of analysis. This is anintroduction to several of the techniques.
There are a variety of scripts available to examine some of the lipidbilayer properties. As an introduction to analysis of the lipid bilayerproperties, this section explains how to obtain the area per lipid andtime-averaged electron density profile for a trajectory.

Area per Lipid

Area per lipid is the average area that a single phospholipid occupiesin an interface and is usually reported in Angstroms squared. It can becalculated with:
Area per lipid = (box X dimension) * (box Y dimension) / (number of phospholipids per layer)
cpptraj is an Amber trajectory analysis program that can perform avariety of actions on trajectory files. In order to calculate the areaper lipid of a lipid bilayer system, you can use this program toextract the periodic box dimensions from a trajectory. In order to runcpptraj, an input file is needed for this operation. A sample inputfile with comments is provided below.
box_dimension.cpptraj
In order to run cpptraj on the trajectory use the command:
The file vector.dat has the box dimensions stored in columns in thefile. However the first two lines of the file is a header that must beremoved for the calculation.
Use tail to strip the first two lines of the file:
To actually calculate the area per lipid as a function of time, the boxx dimension (column 2) must be multiplied by the box y dimension(column 3).
Use awk to do the arithmetic on vector2.tmp:
area_per_lipid.dat can be easily plotted with your favorite plotting tool.

Bales Of Amber Mac Os X

For example, to plot with gnuplot, start gnuplot:
Plot with the gnuplot:
The area per lipid of the bilayer system is seen to converge after some simulation time.

Electron-Density Profile

The electron density profile provides a time-averaged measurement ofthe density of electrons through the lipid bilayer. This can becalculated throughout a trajectory in a fairly straightforward manner.Electron density plots are often compared with experimental models ofthe electron density.
Lipid analysis functions have been added to the cpptraj program.cpptraj is now capable of calculating an electron-density profile andlipid order parameters.
In order to calculate the electron density with cpptraj, an script input file for cpptraj is needed.
A sample input file is provided below:
density.cpptraj
To run the modified ptraj executable script, use:
Now you have a file electron_density.dat with the electron density.
Plot with your favorite program. For example, in the gnuplot command line:


One recently studied membrane-bound protein system is that ofrhodopsin, the visual pigment in photoreceptor cells. Rhodopsin was oneof the first G-Coupled Protein Receptors (GPCR) to be crystallized.Grossfield et al. used Blue Gene, one of the top supercomputers at thetime, to study rhodopsin on extremely long time scales. In particular,the authors were able to examine the dynamics of internal watermolecules in the receptor and their effect on the function of rhodopsin[5].
In this section, a summary of the process to build a starting structureand prepare a molecular dynamics simulation for rhodopsin is outlined.

Build a Rhodopsin 2:2:1 POPC:POPE:Cholesterol System

The crystal structure for rhodopsin is available at the RCSB PDB database, ID 1U19.
For molecular dynamics of this system, it is possible to again use the CHARMM-GUI web site to build the structure.
Select Protein/Membrane Systemfor your membrane-bound protein and enter the RCSB PDB ID of rhodopsinand choose RCSB from the structure type.
Model/Chain Selection Options
You'll need to choose the chain from the structure file. In this case, choose just the chain A for the initial structure.
Continue to the next step.

Note
In a more detailed simulation, theretinal molecule should be included in the simulation. This tutorialwill not cover how to prepare parameters for retinal.
PDB Manipulation Options
In the next step, the PDB must be further processed.
The ACE residue may be renamedto nothing to remove it from the structure. There should be a disulfidebond between cysteine residues 110 and 187.
Continue to the next step.

Computed Energy, Orientation Options, Positioning Options, Area Calculation Options
The rhodopsin structure must now be oriented in the Z direction for the lipid bilayer structure.
For the orientation of the protein, use the Align the First Principal Axis Along z.
Continue to the next step.

Calculated Cross Sectional Area, System Size Determination Options
This page now shows the cross sectional area of the protein within thebilayer. In the Grossfield paper, the authors use a mixture of 2:2:1SDPC:SDPE:Cholesterol.
Because SD is not supported by Lipid14 currently, choose 50:50:25 POPC:POPE:Cholesterol.
Continue to the next step.

Determined System Size, System Building Options, Component Building Options
In the Grossfield paper, the authors used an ionic concentration of 14 sodium ions and 16 potassium ions for their system.
Set the ionic concentration to 0.07 M KCl for this simulation.
Continue with CHARMM-GUI withthe default settings until the completion of step 5. After this, thesystem is assembled and ready to be downloaded.

step5_assembly.pdb
This formatted PDB file can now be processed via the script charmmlipid2amber.py.
Warning
In this example, the CHARMM-GUI does not includea TER card after the protein chain. TER cards ending chains are part ofthe PDB format standard and must be added to the PDB file beforeprocessing with charmmlipid2amber.py.
Once the file has been processed, the next step is to start LEaP. InLEaP, a force field for the protein can be loaded , such as ff12SB along with theLipid14 force field. Then it is possible to load thestructure and build the parameter and topology file in LEaP.
In addition to this tutorial, several other resources are available.Please refer to the following web sites for further information:

• Amber Manuals
  A reference for installing and using Amber software
  
• Amber Tutorials
  Detailed step-by-step guides to specific tasks with Amber software
  
• Amber Mailing List
  An active mailing list with archives since 1999
  

[1] Dickson, C.J., Madej, B.D., Skjevik,A.A., Betz, R.M., Teigen, K., Gould, I.R., Walker, R.C., 'Lipid14: TheAmber Lipid Force Field', J. Chem. Theory Comput., 2014, 10(2), 865-879.
DOI: 10.1021/ct4010307
[2] Skjevik,A.A,; Madej. B.D.; Walker, R.C.; Teigen, K.; 'LIPID11: A ModularFramework for Lipid Simulations Using Amber', Journal of PhysicalChemistry B, 2012, 116 (36), pp 11124-11136.
DOI: 10.1021/jp3059992
[3] Dickson,C.J.; Rosso, L.; Betz, R.M.; Walker, R.C.; Gould, I.R., 'GAFFlipid: aGeneral Amber Force Field for the accurate molecular dynamicssimulation of phospholipid.', Soft Matter, 2012, 8, 9617-9627.
DOI: 10.1039/C2SM26007G
[4] Kucerka, N.; Tristram-Nagle, S; Nagle, J. J. Membrane Biol. 2005, 208, 193-202.
[5] Alan Grossfield, Michael C. Pitman, Scott E. Feller, Olivier Soubias, Klaus Gawrisch,
Internal Hydration Increases during Activation of the G-Protein-Coupled Receptor Rhodopsin,
Journal of Molecular Biology, Volume 381, Issue 2, 29 August 2008, Pages 478-486,
ISSN 0022-2836.