Real Space Structural Real Space Structural Refinement Refinement in EMin EM

Houston Workshop

April 20, 2006

James Z. ChenJames Z. ChenGrigorieff Lab, HHMI at Brandeis University

Topics

Model-density docking

Ligand-receptor docking

Force-field-restrained EM docking

Application demo

Model-Density Docking

SITUS [Wriggers et al., 1999]

COAN [Volkmann & Hanein, 1999]

DOCKEM [Roseman, 2000]

EMFit [Rossmann et al., 2001]

Helixhunter & Foldhunter [Jiang et al., 2001]

RSRef [Chapman, 1995]

Ligand-Receptor Docking

Molecular Mechanics Force FieldsBond, angle, dihedral interactions

VDW, Coulomb, H-bond interactions

Environmental mean-fields

Molecular Interaction SimulationX-PLOR [Brünger, 1987]

DOCK and AutoDOCK [Olson et al., 1990]

VMD, FlexDock, FT-DOCK, and many more

Docking in EM

Why force-fieldsReduce DOF in low-resolution EM density map

Density matching ≠ realistic structure

Molecular complex modelingProtein-ligand docking prediction (CAPRI)

Density-guided homology modelingProtein folding prediction (CASP)

RSRef Methodology

( )2∑ −+=⊕atoms

calcobs bkE ρρ

**

*2**

2)2sin()(4)(

*

*

ddrd

rdddgQrhigh

low

d

dcalc π

ππρ ∫=

A resolution-dependant density calculation

RSMD Refinement

DynamicsDynamics

• Rigid-body MD• Torsion-angle MD• Cartesian-space MD• Energy minimization

E E ETOTAL CHEM= + ⊕.

EM Refinement Protocol

1. Rigid-body annotation

2. NCS restraints enforcement

3. Torsion-angle degree of freedom

4. Refinement via simulated annealing

Helical Structure of Flagella HOOK

Optimal Conformation

Crystal StructureCrystal StructureE(bond)=0.990 E(E(bond)=0.990 E(anglangl)=10.353 E()=10.353 E(torstors)=3.161 )=3.161 E(VDW )=E(VDW )=--208. E(PVDW)=0.3E+8 E(208. E(PVDW)=0.3E+8 E(rresrres)=167.0)=167.0

Manual AdjustmentManual AdjustmentE(bond)=0.975 E(E(bond)=0.975 E(anglangl)=10.397 E()=10.397 E(torstors)=3.169 )=3.169 E(VDW)=0.5E+7 E(PVDW)=0.3E+5 E(E(VDW)=0.5E+7 E(PVDW)=0.3E+5 E(rresrres)=161.0)=161.0

RSMD RefinementRSMD RefinementE(bond)=0.923 E(E(bond)=0.923 E(anglangl)=10.351 E()=10.351 E(torstors)=3.138 )=3.138 E(VDW )=E(VDW )=--191. E(PVDW)=191. E(PVDW)=--14.38 E(14.38 E(rresrres)=156.0)=156.0

Hierarchical Refinement of NSF

20 20 ÅÅ 17 17 ÅÅ

14 14 ÅÅ 11 11 ÅÅ

Actin-DHP Docking

CC = 0.49CC = 0.49 CC = 0.67CC = 0.67

The Road Ahead …

Density map & structureAtomic scattering v.s. electron scattering

Time-resolved dynamics study via EMConformational snapshots from EM experiments

Dynamics simulation to “connect the dots”

Interpretation of an EM refinementResolution, resolution, and resolution !!!

EM modeling from large, heterogeneous dataset