Resolving the Structure of Viral Genomes with Atomic Resolution - - PowerPoint PPT Presentation

Resolving the Structure of Viral Genomes with Atomic Resolution

Aleksei Aksimentiev Department of Physics University of Illinois at Urbana-Champaign

I use Blue Waters to …

… understand molecular underpinnings of life … build biologically inspired systems

DNA, the blueprint

Viral genome, the program of infection

Cryoem reconstruction with concentric rings (Evilevitch et al, UIUC)

http://darwin.bio.uci.edu/~faculty/wagner/hsv2f.html

Herpes virus (HSV)

Open questions:

• What is the 3D structure of the genome?
• How genome ejection is triggered and sustained?
• Can it be used as a drug target?

DNA is a highly charged polymer!

Same sign charges ….

• Same sign charges can

a0ract (in a medium) F F

• Same sign charges repel

(in vacuum) F F

DNA is surrounded by counter ions

+1e, sodium or potassium

+2e, magnesium or calcium +4e,spermine +3e,spermidine

EffecBve a0racBon between DNA is observed when counterions have charge ≥ 2e

All-Atom Molecular Dynamics Simulation of DNA Condensates

Classical Force Field

U(r) = X

bonds

kb(b − b0)2 + X

angles

kθ(θ − θ0)2 )2 + X

dihedrals

kφ(1 + cos(nφ − φ0)) # φ θ b

Bonded parameters from quantum mechanics

}

LJ parameters from experiments

+ X

non-bonded pairs i,j

4✏ij "✓ij rij ◆12 − ✓ij rij ◆6## + X

non-bonded pairs i,j

qiqj 4⇡✏0rij

}

Partial charges from quantum mechanics

Add 64 DNA helices Add polyamine cations (+4) Add 150 mM NaCl Add explicit water Solve the equation of motion (F= ma) under periodic boundary condition in all directions

DNA-confining wall of radius R

Apply a half-harmonic wall potential only to DNA

y (nm)

15-ns MD

Cross-sectional view of MD using CHARMM27

Na

Standard CHARMM & AMBER Force Fields Are Not Perfect for the Simulation of DNA Condensates

x (nm)

1

2 4 6

10

2 4 6

100 Pressure (bar) 30 25 DNA-DNA distance (Å) 1

2 4 6

10

2 4 6

100 Pressure (bar) 32 30 28 26 24 22 DNA-DNA distance (Å)

[Na] = 250 mM [Mg] = 20 mM

Rau et al. Rau et al.

CHARMM27 AMBER99 CHARMM27 AMBER99

Long-lasting contact ion pairs (CIP) between Na+ and phosphate stabilize contact DNA pairs.

2 4

1

2 4

10

2 4

100 Pressure (bar) 30 28 26 24 22 DNA-DNA distance (Å)

[spermine] = 2 mM

Todd et al.

C H A R M M 2 7 AMBER99

Due to excessive CIP formation, the simulations underestimate both inter-DNA distance and pressure in DNA array systems.

[Na] = 250 mM outside

Yoo & Aksimentiev, JPCL 2012

Harmonic wall

Rau et al, PNAS 1984 Todd et al, BJ 2008

Champaign-Urbana Non-Bonded FIX (CUFIX): Improved Lennard-Jones Parameters for CHARMM & AMBER

• “Much of what is known about association and

dissociation of solutes and ions comes from measurements of colligative properties” — Molecular driving forces by Dill & Bromberg.

• 4
• 2

2 4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Density (g/cm

3)

Na Acetate Water Total

Na

≈

Dimethylphosphate Acetate

Yoo & Aksimentiev, JPCL 2012 Yoo & Aksimentiev, JCTC 2016 Yoo, Wilson & Aksimentiev, Biopolymers 2016

200 150 100 50 OSM pressure (bar) 4 3 2 1 molal conc (m) 200 150 100 50 4 3 2 1

Murad & Powles, JCP 1993 Luo & Roux, JPCL 2010

0.20 0.15 0.10 0.05 0.00

• 0.05
• 0.10

ULJ (kcal/mol) 4.0 3.6 3.2 2.8 Na–O distance (Å) 0.20 0.15 0.10 0.05 0.00

• 0.05
• 0.10

4.0 3.6 3.2 2.8

Standard rmin = 3.11 Å rmin = 3.20 Å Standard NBFIX

CUFIX for CHARMM36 & AMBER99

Effectively infinite slab under PBC

• Exp. from Robinson 1959

http://bionano.physics.illinois.edu/CUFIX

Yoo & Aksimentiev, JPCL 2012

1

2 4 6

10

2 4 6

100 Pressure (bar) 32 30 28 26 24 22 DNA-DNA distance (Å) 1

2 4 6

10

2 4 6

100 Pressure (bar) 30 25 DNA-DNA distance (Å)

CUFIX Improves Simulations of DNA Condensates

• 120
• 80
• 40

40 80 120 y (Å)

• 120 -80
• 40

40 80 120 x (Å)

• 120
• 80
• 40

40 80 120 y (Å)

• 120 -80
• 40

40 80 120 x (Å)

• 120
• 80
• 40

40 80 120 y (Å)

• 120 -80
• 40

40 80 120 x (Å)

[Na] = 250 mM [Mg] = 20 mM [spermine] = 2 mM

AMBER99 AMBER99

2 4

1

2 4

10

2 4

100 Pressure (bar) 30 28 26 24 22 DNA-DNA distance (Å)

A M B E R 9 9

A M B E R 9 9 + C U F I X AMBER99 + CUFIX

Yoo & Aksimentiev, NAR 2016

A M B E R 9 9 + C U F I X ( M D i n c l u d e d 2 m M N a )

CHARMM27 CHARMM27 CHARMM27

DNA is packaged by a motor

At higher forces, DNA will deform Packaging process is slow (~min), all-atom simulation at physiological forces is not possible Can one simulate the process? Takes about 3 minutes to pack DNA 130 times longer than the capsid ! Max Force: 100pN Movie: Carlos Bustamante Lab

Strategy: change resolution for speed and detail

Strategy: change resolution for speed and detail

Strategy: change resolution for speed and detail

Strategy: change resolution for speed and detail

Strategy: change resolution for speed and detail

Strategy: change resolution for speed and detail

500 bp dsDNA fragment modeled at different resolutions

24 bp/2 beads 12 bp/2 beads 6 bp/2 beads 3 bp/2 beads 1 bp/2 beads All-atom, ~100 bp

Interactions in a simple coarse-grained DNA model

Interactions in a simple coarse-grained DNA model

Bond potential

r0 = nbp × 3.4 ˚ A f0 = 1000pN kspring = f0/r0 Elastic constant r0

Force Extension

http://www.phys.ens.fr/~cocco/Art/24physworld.pdf

Interactions in a simple coarse-grained DNA model

Angle potential

Persistence length s

Lp = 50 nm

e−s/Lp = ⟨cos θ⟩ = ∫𝕐 d𝕐 cos θ δ(θ′[𝕐] − θ)e−βU[𝕐] ∫𝕐 d𝕐 e−βU[𝕐] = ∫

π 0 sin θ dθ cos θe−β 1

2 kspringθ2

∫

π 0 sin θ dθ e−β 1

2 kspringθ2

Interactions in a simple coarse-grained DNA model

90°

φ

Dihedral angle potential

Twist persistence length Ltw = 90 nm hcos φi = es/Ltw Z π dφ cos φ e

kdihed(φ−φ0)2 2kBT

= es/Ltw φ0 = s ⇥ 10.14/˚ A

Interactions in a simple coarse-grained DNA model

4 nm
 cutoff

z Periodic in axial axis

Pr

Interactions in a simple coarse-grained DNA model

4 nm
 cutoff

80 80 4 bp/

Optimized to reproduce Rau & Parsegian pressure Half-harmonic
 wall to prevent
 strand crossing

Mapping between coarse-grained resolutions

For each helix, fit a 3D spline through bead coordinates at end of simulation

coordinate 1D spline

Fit a spline between
 quaternion representation of rotations

Packaging viruses with ARBD

ARBD: Atomic Resolution Brownian Dynamics (multi-resolution)

Package DNA (CG) with ARBD, into CryoEM reconstruction of a HK97 bacteriophage capsid. A cryoEM map of the portal is fitted into the original capsid reconstruction, and DNA is packaged through the portal.

Smooth, purely repulsive grid-based potential obtained by blurring cryoEM density and adding the portal

MulB-resoluBon packaging dsDNA viruses

Internal pressure during packaging

27

Pressure (atm) Percent of DNA

• utside capsid, %

Evilevitch et al, PNAS

Comparison to structural data

28

Simulation Experiment

• J. Mol. Biol. (2009) 391, 471-483, Hendrix et al

Cryo-electron microscopy q [Å−1]

experiment simulation

I(q) [a . u.] Small Angle X-ray Scattering

Experiment: Journal of molecular biology, 408: 541 (2011)

Simulation SAXS data were generated from CRYSOL, using an atomistic PDB of the protein coat and packaged DNA

Conclusions and outlook

29

Obtained first atomic-resolution structure of packaged viral particle Developed accurate multi-resolution representation of DNA—DNA and DNA—protein interactions To do: Extend the model to ssRNA and ssDNA viruses

Acknowledgements

• Funding through CPLC
• Computations

Jejoong Yoo David Winogradoff Chris Maffeo Kush Coshic