http://im.bioinformatics.ku.edu/Publications/download/Membrane_Builder.

pdf
updated on May 13, 2012

Membrane Protein Simulation:

One Step Closer to Users
Background, Present, and Future of CHARMM-GUI Membrane Builder

Wonpil Im
Department of Molecular Biosciences and
Center for Bioinformatics
University of Kansas

http://im.bioinformatics.ku.edu

http://www.charmm-gui.org

http://www.glycanstructure.org

Ion Channels

KcsA K+ channel

G-Protein Coupled Receptor

(GPCR)

rhodopsin

Ion Channels

G-Protein Coupled Receptor

(GPCR)

Membrane proteins play important roles in many cellular processes

: signaling, transduction of energy, and transport of ions and small molecules

Association/Dissociation of Transmembrane helices

: fundamental process in membrane protein structural biology

KcsAclass
K+ channel
The largest
of drug target

rhodopsin

: 2030% of the protein-encoding regions of known genomes encode

lipids, membranes, proteins

Golgi Apparatus!

Endoplasmic !
Reticulum!

Nucleus!

Mitochondria!

Good Fences make Good Neighbors

- Robert Frost, 1914 -

http://www.usd.edu/~bgoodman/ReviewFrames.htm!

Amusing, but
Golebiewska, Kay, Masters, Grinstein, Im, Pastor, Scarlata, McLaughlin (2011) Mol. Biol. Cell 22:3498-3507

Hi guys,"
"
This little movie might amuse you. Rich and Wonpil: "
Dictyostelium is a slime mold or social amoeba. It lives alone when the living is easy, "
but when it gets cold and dark, it huddles together, forming slugs and fruiting bodies. "
In this example, a lone guy is trying to eat "
a yeast that is just too long."
"
"
PIP2 in green, actin in red."
"
"
Cheers"
Stuart"

Ion Channels

KcsA K+ channel

G-Protein Coupled Receptor

(GPCR)

rhodopsin

Membrane Protein:

realistic atomic simulation

Im & Roux (2002) J. Mol. Biol.

How to Build / Simulate / Analyze

Membrane Systems

Molecular Force Field

: what types of lipid molecules are available? Are they good enough?
System Building / Simulation Protocols
: why do we care?
CHARMM-GUI Membrane Builder
: background, present, and future

CHARMM Force Field

Topology Files: !

Parameter Files:!!

!top_all22_prot.rtf!

!par_all22_prot.prm!

!top_all27_na.rtf!

!par_all27_na.prm!

!top_all36_carb.rtf!

!par_all36_carb.prm!

!top_all36_lipid.rtf!

!par_all36_lipid.prm!

!top_all36_cgenff.rtf!

!par_all36_cgenff.prm!

Topology/Parameter Stream Files: !

from CHARMM-GUI!

!toppar_water_ions.str!
!toppar_all36_lipid_cholesterol.str!
!toppar_all36_glycopeptide.str!
!toppar_all22_prot_fluoro_alkanes.str!
!toppar_all22_prot_aliphatic_c27.str!

- toppar.str!
reading all top & par files!

- toppar directory!

Lipid Molecules: Glycerophospholipids

R = choline, serine, ethanolamine, inositol
lipid head groups
(hydrophilic)
C3
C2

L=S

C1

1
2

lipid tails
(hydrophobic)

Fatty acids:
saturated
monounsaturated with one double bond
polyunsaturated with multiple double bonds

Some Natural Fatty Acids

Chain length
Saturation
Solubility
Melting point

CHARMM C36 Lipid Force Field

CHARMM C36 Lipid Force Field

POPC
1-palmitoyl2-oleoyl
phosphatidylcholine

CHARMM C36 Lipid Force Field

SAPC
1-stearoyl2-arachidonoyl
phosphatidylcholine

CHARMM C36 Lipid Force Field

Klauda, Venable, Freites, OConnor, Tobias, Mondragon-Ramires, Vorobyov, MacKerell and Pastor (2010) J. Phys. Chem. B 114, 78307843

CHARMM C36 Lipid Force Field

Acyl Chain Order Parameter

SCD

1
= 3cos2 ! CH !1
2

CH is the angle between

the CH bond vector and
the bilayer normal

How to Build & Simulate

the Membrane Systems?

How to Build and Simulate?

How to Build and Simulate?

Assembly of uncorrelated pieces!!
!
!
Equilibration, Equilibration, and Equilibration!

Three Ensembles of Interest!

NPT : !constant pressure!
!temperature!
NPAT : constant pressure!
!surface area (along XY)!
!temperature!
NPT : constant pressure!
!surface tension!
!temperature!

Overview of Membrane Builder

Jo, Kim, and Im (2007) PLoS ONE 2:e880
Jo, Lim, Klauda, and Im (2009) Biophys. J. 97:50

CHARMM-GUI!

Membrane Builder!

Overview of Membrane Builder

Jo, Kim, and Im (2007) PLoS ONE 2:e880
Jo, Lim, Klauda, and Im (2009) Biophys. J. 97:50

Protein/Bilayer

Lipid Bilayer Only

Pure or Mixed Lipid Bilayer Systems!

: 35 lipid types"
"
Optimized CHARMM & NAMD inputs!
: for both equilibration and production"
"
Flexible System Size Determination!
: Insertion or Replacement Methods"

NAMD inputs files (v2.7b3 or after) can be found

in the "namd" directory when you download a tar
archive ("charmm-gui.tgz") after all the input file
generation.

Membrane Builder: STEP 1

Jo, Kim, and Im (2007) PLoS ONE 2:e880
Jo, Lim, Klauda, and Im (2009) Biophys. J. 97:50

Protein/Bilayer

Lipid Bilayer Only

Simple protein structure modifications (e.g.,

disulfide bond generation, terminal group changes,
phosphorylation, and protonation) can also be made
during this step.
The protein must be oriented with respect to a
membrane bilayer whose normal is parallel to the
Z-axis and whose center is located at Z=0.
RCSB PDB structures are NOT pre-oriented, but
can be oriented in STEP 2.
OPM (http://opm.phar.umich.edu) provide preoriented protein coordinates with respect to the
bilayer.

Membrane Builder: STEP 2

Jo, Kim, and Im (2007) PLoS ONE 2:e880
Jo, Lim, Klauda, and Im (2009) Biophys. J. 97:50

Protein/Bilayer

Lipid Bilayer Only

Membrane Builder: STEP 2

Jo, Kim, and Im (2007) PLoS ONE 2:e880
Jo, Lim, Klauda, and Im (2009) Biophys. J. 97:50

Protein/Bilayer

Lipid Bilayer Only

Protein Cross-Sectional Area!

Membrane Builder: STEP 3

Jo, Kim, and Im (2007) PLoS ONE 2:e880
Jo, Lim, Klauda, and Im (2009) Biophys. J. 97:50

Protein/Bilayer

Lipid Bilayer Only

THIS IS THE MOST CRITICAL STEP!

Initial system size in XY:
( total lipid area + protein area )1/2
Initial system size in Z:
protein extent in Z + water thickness x 2
Selection of homo- or heterogeneous bilayer
hetero-option can be used for homogeneous
bilayer generation

Membrane Builder: STEP 3

Jo, Kim, and Im (2007) PLoS ONE 2:e880
Jo, Lim, Klauda, and Im (2009) Biophys. J. 97:50

Protein/Bilayer

Lipid Bilayer Only

Homogeneous Bilayer Case!

Membrane Builder: STEP 3

Jo, Kim, and Im (2007) PLoS ONE 2:e880
Jo, Lim, Klauda, and Im (2009) Biophys. J. 97:50

Protein/Bilayer

Lipid Bilayer Only

Homogeneous Bilayer Case!

Membrane Builder: STEP 3

Jo, Kim, and Im (2007) PLoS ONE 2:e880
Jo, Lim, Klauda, and Im (2009) Biophys. J. 97:50

Protein/Bilayer

Lipid Bilayer Only

Heterogeneous Bilayer Case!

Membrane Builder: STEP 3

Jo, Kim, and Im (2007) PLoS ONE 2:e880
Jo, Lim, Klauda, and Im (2009) Biophys. J. 97:50

Protein/Bilayer

Lipid Bilayer Only

Heterogeneous Bilayer Case!

Membrane Builder: STEP 3

Jo, Kim, and Im (2007) PLoS ONE 2:e880
Jo, Lim, Klauda, and Im (2009) Biophys. J. 97:50

Protein/Bilayer

Lipid Bilayer Only

Heterogeneous Bilayer Case!

Membrane Builder: STEP 3

Jo, Kim, and Im (2007) PLoS ONE 2:e880
Jo, Lim, Klauda, and Im (2009) Biophys. J. 97:50

Protein/Bilayer

Lipid Bilayer Only

step3_packing.pdb!

+ image atoms!
Please have a look at it!!

Membrane Builder: STEP 4

Jo, Kim, and Im (2007) PLoS ONE 2:e880
Jo, Lim, Klauda, and Im (2009) Biophys. J. 97:50

Protein/Bilayer

Lipid Bilayer Only

Membrane Builder: STEP 4

Jo, Kim, and Im (2007) PLoS ONE 2:e880
Jo, Lim, Klauda, and Im (2009) Biophys. J. 97:50

Protein/Bilayer

Lipid Bilayer Only

Lipid Generation: Insertion Method!

Membrane Builder: STEP 4

Jo, Kim, and Im (2007) PLoS ONE 2:e880
Jo, Lim, Klauda, and Im (2009) Biophys. J. 97:50

Protein/Bilayer

Lipid Bilayer Only

Lipid Generation: Insertion Method!

We have pre-generated bilayers with

various hole sizes for each lipid.!

Membrane Builder: STEP 4

Jo, Kim, and Im (2007) PLoS ONE 2:e880
Jo, Lim, Klauda, and Im (2009) Biophys. J. 97:50

Protein/Bilayer

Lipid Bilayer Only

Lipid Generation: Insertion Method!

Membrane Builder: STEP 4

Jo, Kim, and Im (2007) PLoS ONE 2:e880
Jo, Lim, Klauda, and Im (2009) Biophys. J. 97:50

Protein/Bilayer

Lipid Bilayer Only

Lipid Generation: Replacement Method!

These pseudo-lipid spheres were generated in STEP3.!

Membrane Builder: STEP 4

Jo, Kim, and Im (2007) PLoS ONE 2:e880
Jo, Lim, Klauda, and Im (2009) Biophys. J. 97:50

Protein/Bilayer

Lipid Bilayer Only

Lipid Generation: Replacement Method!

We have generated 2000 conformationlibrary for each lipid.!

Membrane Builder: STEP 4

Jo, Kim, and Im (2007) PLoS ONE 2:e880
Jo, Lim, Klauda, and Im (2009) Biophys. J. 97:50

Protein/Bilayer

Lipid Bilayer Only

Lipid Generation!
[step4.1_lipid.inp]!

Water Generation: a slab of water box!

[step4.2_waterbox.inp]!
[step4.4_pwat.inp]!

Ion Generation: Monte-Carlo Runs!

[step4.3_ion.inp]!

van der Waals + 80-scaled Coulombic interactions!

Membrane Builder: STEP 5

Jo, Kim, and Im (2007) PLoS ONE 2:e880
Jo, Lim, Klauda, and Im (2009) Biophys. J. 97:50

Protein/Bilayer

Lipid Bilayer Only

ASSEMBLY

Membrane Builder: STEP 6

Jo, Kim, and Im (2007) PLoS ONE 2:e880
Jo, Lim, Klauda, and Im (2009) Biophys. J. 97:50

Protein/Bilayer

Lipid Bilayer Only

Membrane Builder: STEP 6

Jo, Kim, and Im (2007) PLoS ONE 2:e880
Jo, Lim, Klauda, and Im (2009) Biophys. J. 97:50

Protein/Bilayer

Lipid Bilayer Only

Membrane Builder: STEP 7

Jo, Kim, and Im (2007) PLoS ONE 2:e880
Jo, Lim, Klauda, and Im (2009) Biophys. J. 97:50

Protein/Bilayer

Lipid Bilayer Only

Production Run without Restraints:!

There is an input for STEP 7!

Hopefully in the near future

if we get good resources
you may see

Different Membranes

Monolayers

Micelles

Vesicles

More Lipid Types: Three Cases

PIP2

cardiolipin

lipopolysaccharide

Lipopolysaccharide Membranes

10 25 repeating units
each unit with 3-6 linear or branched sugars
highly variable

Lipopolysaccharide Membranes
Jo, Wu, Stuhlsatz, Klauda, MacKerell, Widmalm, and Im (2012)
Methods in Molecular Biology: Glycoinformatics (submitted)

Membrane Analyzer
Have you ever imagined a web-based toolset for !
membrane simulation trajectory analysis?!

Updates on CHARMM-GUI

CHARMM-GUI Development Project

Jo, Kim, Iyer, and Im (2008) J. Comput. Chem. 29:1859

CHARMM Input Generator

PDB Reader"

Solvator"

Quick MD Simulator"

Implicit Solvent Modeller"

Targeted/Steered/Pulling "
MD Simulator"

Membrane Builder"

Path Finder"

Solvent Boundary Potential"

Utilizer"

QM/MM Simulator"

PBEQ Solver"

Free Energy Calculator"

Normal Mode Analyzer"

REX-MD Simulator"

NMR Structure Calculator"

EMAP Utilizer"

Glycan Reader"

Ligand Reader"

available now"
work in progress"
future plan "

PBEQ Solver
Jo, Vargyas,Vasko-Szedlar, Roux, and Im (2008) Nucl. Acids Res. 36:W270

Online Visualization!

Glycan Simulation

Glycan = Carbohydrate = Sugar = Saccharide

Basic structure of
carbohydrate

Carbohydrate can
be linear / cyclic

Two isomeric ring

can be found

Saccharide units can be covalently linked to form larger structure

sucrose (table sugar)

lactose

amylose

Why Glycan?

Gal
GlcNAc

Neu5Ac
Man

Fuc

Why Glycan?
1. Glycans modulate protein structure, dynamics, and function
2. Glycans are related to vaccine developments
3. There are critical needs for a general tool for glycan modeling and simulation

Glycans have impacts on inflammatory activity of Immunoglobin IgG1

anti-inflammatory

pro-inflammatory

16
14

16

12

Fc receptor binding site

13
14

14

14

12

Gal
GlcNAc

Neu5Ac
Man

Fuc

Why Glycan?
1. Glycans modulate protein structure, dynamics, and function
2. Glycans are related to vaccine developments
3. There are critical needs for a general tool for glycan modeling and simulation

HIV gp120 envelop protein has lots of glycans, about 50% of the total mass

Trimeric spike

Why Glycan?
1. Glycans modulate protein structure, dynamics, and function
2. Glycans are related to vaccine developments
3. There are critical needs for a general tool for glycan modeling and simulation

Glycans make a CHARMM input less charming

1. Unlike the same peptide bond in protein,

carbohydrates in a glycan chain are connected by
different glycosidic linkages.
2. 30% of carbohydrate residue names in PDB are
WRONG.
3. Glycan structures in PDB are often incomplete.

Why Glycan?
1. Glycans modulate protein structure, dynamics, and function
2. Glycans are related to vaccine developments
3. There are critical needs for a general tool for glycan modeling and simulation

Glycans make a CHARMM input less charming

! Read CARA
read sequence card unit 5
* Glycan Chain CARA: PDB chain
*
10
AMAN BMAN BGLCNA BFUC BGLCNA
AMAN BGLCNA BGAL BGAL BGLCNA
generate CARA first none last none setup
patch 13AB CARA 2 CARA 1
patch 14BA CARA 3 CARA 2
patch 14BA CARA 5 CARA 3
patch 16BB CARA 5 CARA 4
patch NGLB PROA 297 CARA 5 ! patch to protein
patch 16AB CARA 2 CARA 6
patch 12BB CARA 6 CARA 7
patch 14BA CARA 7 CARA 8
patch 14BA CARA 10 CARA 9
patch 12BB CARA 1 CARA 10
autogenerate angle dihe

Glycan Reader
Jo, Song, Desaire, MacKerell, and Im (2011) J. Comput. Chem. 32:3135-3141

(A- B) automatic detection of

carbohydrate molecules
using graph representation

Glycan Reader
Jo, Song, Desaire, MacKerell, and Im (2011) J. Comput. Chem. 32:3135-3141

(A- B) automatic detection of

carbohydrate molecules
using graph representation
(B-C) automatic annotation of
carbohydrates based on their
3D structures

Glycan Reader
Jo, Song, Desaire, MacKerell, and Im (2011) J. Comput. Chem. 32:3135-3141

(A- B) automatic detection of

carbohydrate molecules
using graph representation
(B-C) automatic annotation of
carbohydrates based on their
3D structures
(C-D) automatic recognition of
glycosidic linkages between
(E) automatic generation of CHARMM inputs
carbohydrates as well as N-/Oglycosidic linkages to proteins

Glycan Reader
Jo, Song, Desaire, MacKerell, and Im (2011) J. Comput. Chem. 32:3135-3141

cyclodextrin

Glycan Reader
Jo, Song, Desaire, MacKerell, and Im (2011) J. Comput. Chem. 32:3135-3141

Glycan Reader
Jo, Song, Desaire, MacKerell, and Im (2011) J. Comput. Chem. 32:3135-3141

Ligand Binding Free Energy

Ligand Binding Free Energy Calculation

electrostatic
dispersive
replusive

Ligand Binding Free Energy Calculation

Binding

+

Find right orientation

Close contact

Rearrangement

Binding

Desolvation

Ligand Binding Free Energy Calculation

Deng and Roux, J. Chem. Theory Comput. (2006)

Deng and Roux, J. Phys. Chem. B (2006)

Ligand Binder
Jo, Jiang, Lee, Roux, and Im (2012) submitted

Ligand Binder
Jo, Jiang, Lee, Roux, and Im (2012) submitted

Ligand Binder
Jo, Jiang, Lee, Roux, and Im (2012) submitted

Series of inputs will be generated to setup boundary potential,

equilibrate, and simulate free energy.
Input files are ready to run in a local cluster.
Free energy simulation typically takes 12 hrs to 20 hrs with ~130 CPUs.

Ligand Binder
Jo, Jiang, Lee, Roux, and Im (2012) submitted

Step1:

PDB Reading

Step 2:

Solvation

Step 3:

Boundary
Potential

Step 4:

Equilibration

Site

Step 5:

Free Energy
Simulation

GSBP

Step 3:

Boundary

Pontential

Step 2:

Solvation

Bulk

Step 6:

WHAM

Buffer
Implicit

SSBP

Benzene

T4 Lysozyme

PDB: 181L

Implicit

Explicit

Ligand Binder
Jo, Jiang, Lee, Roux, and Im (2012) submitted

Step1:

PDB Reading

STEP1

Input
Output

STEP2

Input
Output

STEP3

Input

Output

Step 2:

Solvation

Step 3:

Boundary
Potential

Step 4:

Equilibration

Step 5:

Free Energy
Simulation

Filename
step1_pdbreader.inp

Description
Input files for structure initialization

step1_pdbreader.pdb
@lig/@lig.rtf
@lig/@lig_g.rtf
@lig/@lig.prm
@lig/ndihe.stsr
step2.1_site_solvator.inp
step2.2_bulk_solvator.inp
step2.1_site_solvator.pdb
step2.2_bulk_solvator.pdb
step3.1_gsbp_setup.inp
step3.2_ssbp_setup.inp
config.py
step3_job.pbs
step3.1_gsbp_setup.pdb
step3.1_gsbp_setup.mij
step3.1_gsbp_setup.phix
step3.2_ssbp_setup.pdb

Structure after initialization in CHARMM

Ligand topology file generated
Ligand topology file (with atom groups)
Ligand parameter file generated
Symmetric groups found in the ligand
Solvate the binding site with water sphere
Solvate the ligand with water sphere
Solvated simulation system of binding site
Solvated simulation system of bulk solvent
Setup GSBP simulation system for SITE system
Setup SSBP simulation system for BULK system
Contains several configuration variables
Example PBS script for batch system
SITE simulation system after GSBP setup
Calculated multipolar reaction field
Calculated external reaction field
BULK simulation system after SSBP setup

Step 6:

WHAM

Ligand Binder
Jo, Jiang, Lee, Roux, and Im (2012) submitted

STEP4

Input

Output

STEP5

Input

STEP6

Input

Filename
step4.1_site_gcmc.inp
step4.2_site_equil.inp
step4.3_restraint_setup.inp
step4.3_restraint_geo.str
step4.3_restraint_geotest.str
step4.3_restraint_ref.str
step4.4_bulk_equil.inp
step4_job.pbs
step4.1_site_gcmc.crd
step4.2_site_equil.dcd
step4.2_site_equil.crd
step4.3_restraint_geo.prm
step4.3_restraint_geo.pdb
step4.3_restraint_ref.prm
step4.3_restraint_consdihe.str
step4.3_restraint_ligave.pdb
step4.4_bulk_equil.pdb
step5.1_site_fes.inp
step5.1_site_gconst.str
step5.2_bulk_fes.inp
step5_jobmanager.com
step5_template.pbs

Description
Adjust the number of water molecules in GSBP
Equilibration of SITE system
Setup anchoring atoms and restraint parameters
Determine anchoring atoms
Test validity of anchoring atoms
Determine the restraint parameters
Equilibration of BULK system
Example PBS script for batch system
SITE system after adjustment of number of water
Trajectory of equilibration of SITE system
Last snapshot of equilibrated SITE system
Anchoring atom definition
Anchoring atoms (named as DUM)
Reference values for restraint potential
Symmetric group restraints
Average structure of bound ligand during equilibration
Last snapshot of equilibrated BULK system
FEP/MD input for SITE system (including PMF input)
Restraints setup for orientation/translation restriction
FEP/MD input for BULK system (including PMF input)
Script for submitting batch jobs
Example PBS script for batch system

step6.1_wham_fep.inp
step6.1_wham_fep.pbs
step6.2_wham_rmsd.inp
step6.2_wham_rmsd.pbs
step6_wham.com
step6_table.py

WHAM calculation input for FEP/MD simulations

Example PBS script for batch system
WHAM calculation input for PMF simulations
Example PBS script for batch system
Script for WHAM calculation
Example script for tabulate the binding free energy

Ion Channel BD Simulation

BD System of OmpF Porin

Buffer 2

GCMC/BD simulation
Total time 1.5 ns
15 ps/frame
150 mV
1 M [KCl]
K+
Cl-

Buffer 1

Im and Roux (2002) J. Mol. Biol. 322:851-869

Im, Seefeld, and Roux (2000) Biophys. J. 79:788-801

BD System of OmpF Porin

symmetric
solutions

Itot
IK
ICl
asymmetric
solutions

Im and Roux (2002) J. Mol. Biol. 322:851-869

Im, Seefeld, and Roux (2000) Biophys. J. 79:788-801

GCMC/BD Ion Simulator

Lee, Jo, Rui, Egwolf, Roux, Pastor, and Im (2012) J. Comput. Chem. 33:331-339

GCMC/BD Ion Simulator

Lee, Jo, Rui, Egwolf, Roux, Pastor, and Im (2012) J. Comput. Chem. 33:331-339

PG-1 Oligomer

VDAC

-Hemolysine

Anthrax PA-pore

Acknowledgment
Group Members

Collaborators

postdoc

graduate

undergraduate

Dr. Kyu Il Lee

Huan Rui

Kevin C. Song

Dr. Soohyung Park

Sunhwan Jo

Danielle Stuhlsatz

Dr. Huisun Lee

Xi Cheng

John Kim

Dr. Emilia Wu

Andrew Beaven

Phillip Morris
Jacob Wilson

Dr. Jinhyuk Lee

Dr. Taehoon Kim

Vidyashankar Iyer

Dr. Thenmalar Rathinavelan

Dr. Roberto De Guzman

Dr. Mario Rivera
Dr. Robert Dunn
Dr. Heather Desaire
Dr. Rich Pastor (NIH)
Dr. Jeff Klauda (UMD)
Dr. Stanley Opella (UCSD)
Dr. Francesca Marassi (Burnham)

The University of Kansas

Dr. Roger Koeppe (U Arkansas)

Alfred P. Sloan Fellowship

Dr. Hyun-Suk Lim (Indiana)

KU HBC JR & Inez Jay Fund

Kansas COBRE-PSF Pilot Project
NSF MCB-0918374
NIH R01 GM092950
HFSP
TeraGrid & ANTON

Dr. Olaf S. Andersen (Cornell)

Dr. Tony Kossiakoff (UC)
Dr. Jose Villadangos (WEHI)
Dr. Matthew Call (WEHI)
Dr. Satoshi Ishido (RIKEN)
Dr. Stuart McLaughlin (Stony Brook)