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SingleSingle--Particle Electron MicroscopyParticle Electron Microscopy
EM EM StructuralStructural AnalysisAnalysis
Quantitative Biology Lectures, Part III
January 30, 2008
James Z. ChenJames Z. Chen
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Electron Microscope
Transmission Electron Microscopy Transmission Electron Microscopy (TEM)(TEM)
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TEM Image Formation
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Specimen Preparation: Negative StainingNegative Staining
++ quick to makequick to make
++ high dosehigh dose --tolerancetolerance
-- preferred orientationpreferred orientation
-- lower resolution (nm)lower resolution (nm)
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Specimen Preparation: CryoCryo--plungingplunging
++ higher resolution (higher resolution ( ÅÅ))
-- low doselow dose --tolerancetolerance
-- must work under LNmust work under LN 22
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•• emulsion filmemulsion film
•• CCDCCD
•• pixel detectorpixel detector
Image Acquisition
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Data Processing Pipeline
Particle Selection
Particle Alignment
Particle Classification
Model Reconstruction
Model Refinement
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Particle Alignment
2D Alignment2D Alignment
•• TranslationTranslation
•• RotationRotation
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Particle Classification
Goal: improve SNR by class averagingGoal: improve SNR by class averaging
•• PCA to identify PCA to identify eigeneigenimages (modes)images (modes)
•• decompose images in thedecompose images in theeigeneigen--spacespace
•• find point clusters find point clusters ⇒⇒⇒⇒⇒⇒⇒⇒ classesclasses
Feature 1Feature 1
Feature 2Feature 2
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Model Reconstruction
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Topics
� Molecular Mechanics Force-Fields
� Docking via MD simulation
� Structure refinement/prediction
� What will be?
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M-M Force-Fields
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MD Simulation
For each atom:For each atom:
ElectrophoreticallyElectrophoretically--driven translocation of a DNA strand through driven translocation of a DNA strand through the transmembrane pore of alphathe transmembrane pore of alpha--hemolysinhemolysin. TCB, UIUC.. TCB, UIUC.
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PDB-Map Docking
Why force-fields?
Stereo-chemically correct structure
Reduce DOF in low-resolution EM density map
Structural analysis / interpretation
Protein/complex structure refinement
EM structure prediction
Molecular conformation prediction
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Restrained MD Refinement
DynamicsDynamics
• Rigid-body MD
• Torsion-angle MD
• Cartesian-space MD
• Energy minimization
E E ETOTAL CHEM= + ⊕.
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Refinement Methodology
( )2
∑ −+=⊕atoms
calcobs bkE ρρ
**
*2**
2
)2sin()(4)(
*
*
ddrd
rdddgQr
high
low
d
d
calc πππρ ∫=
A resolution-dependant density calculation
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Refinement Protocol
1. Rigid-body annotation
2. Torsion-angle degree of freedom
3. Symmetry enforcement (optional)
4. Refinement via simulated annealing
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Actin-DHP Docking
CC = 0.49CC = 0.49 CC = 0.67CC = 0.67
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Helical Structure of Flagella Hook
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Optimal Conformation
Crystal StructureCrystal Structure
E(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 Adjustment
E(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 Refinement
E(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
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TMV Conformational Change
By courtesy ofBy courtesy ofCarsten SachesCarsten Saches..
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Rotavirus TLPRotavirus TLP
�� dsRNA virusdsRNA virus
�� 11 RNA segments11 RNA segments
�� tripletriple --layered capsidlayered capsid
�� 6 structural proteins6 structural proteins
�� diameter ~800 diameter ~800 ÅÅ
�� weight ~60 MDweight ~60 MD
Jayaram et al, Virus Research, 101:67-8, 2004.
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CryoCryo--EM Structural AnalysisEM Structural Analysis
•• TF30: TF30: 300300kVkV
•• dose: 15 dose: 15 –– 20 e20 e--//ÅÅ22
•• defocus: 1.0 defocus: 1.0 –– 3.0 3.0 µµmm
•• ~ 5~ 5,,000 single particles000 single particles
•• projection matchingprojection matching
•• CTF correctionCTF correction
•• FREALIGN refinementFREALIGN refinement
•• resolution: ~ resolution: ~ 44ÅÅ
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EM Density MapEM Density Map
VP6 + VP7VP6 + VP7 αααααααα--helixhelix
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The Road Ahead …
� High-resolution single-particle EM
� Tackle dynamic, heterogeneous systems
� EM & computational structural biology
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Conclusion
Quantitative BiologyQuantitative Biology
Biology
Physics
Mathematics
Computer Science
…