STRUCTURE
DETERMINATION USING
ELECTRON MICROSCOPY
Molecular View of Anatomy: Infectious Diseases
Rutgers Honors Program Seminar, Spring 2011
cathy.lawson@rutgers.edu
ELECTRON MICROSOPY (EM)

EM is a well-established imaging
technique that generates highresolution 2D images of very small
objects.

This lecture provides an overview
of EM as a tool to investigate the
3D shapes of medium to large
macromolecular assemblies.
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GROWTH OF EM METHOD FOR DETERMINING
STRUCTURES OF MACROMOLECULAR ASSEMBLIES
**2010 update**
EMDB: 983 maps
PDB: 358 models
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FROM SAMPLE TO STRUCTURE
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SOME COMMON TERMS
Cryo EM = biological samples are preserved in
vitreous ice and imaged by EM at cryogenic
temperatures.
 Single Particle Reconstruction = 3D map
generated by averaging 2D images of many
individual specimens in multiple orientations.
 Tomogram = 3D map of a single specimen
generated by integrating multiple images of the
specimen taken at different angles.

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SAMPLE TYPES
Type
Examples
Reconstruction
Method
Typical
Resolution
Single
particles
Icosahedral viruses,
GroEL, ribosome
Single particle
reconstruction
20-3 Å
Filaments
Flagella, filamentous
viruses, actin, tubular
crystals
Helical
reconstruction
15-3 Å
2D crystals
Catalase, aquaporin,
tubulin
Electron
crystallography
10-2 Å
Ensembles
HIV capsids, cell
cytoskeleton
Electron
Tomography
40-20 Å
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SINGLE PARTICLES
•
MW lower limit: ~200 kDa
Virus
Ribosome
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Image sources: EMDB, Joachim Frank, Sacha De Carlo
SINGLE PARTICLES—POINT SYMMETRY
Cyclic
Octahedral
Dihedral
Tetrahedral
Icosahedral
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FILAMENTS
Acetylcholine receptor tubular crystal
Tobacco Mosaic Virus
Image: gingi.uchicago.edu/achr.html
Actin-myosin complex
Image: ami.scripps.edu
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HIGH RESOLUTION—SINGLE
PARTICLE, FILAMENT (4-5 Å)
GroEL
Tobacco Mosaic Virus
Ludtke et al (2008)
Structure 16: 441-448.
Sachse et al (2007)
JMB 371: 812-835.
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2D CRYSTALS
Aquaporin 2D crystals, electron diffraction
Lipid-protein interactions
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Gonen et al. 2005. Nature 438:633-8.
ENSEMBLES
Reconstruction of HIV capsids by cryoEM tomography
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Benjamin et al. 2005. J. Mol Biol. 346:577-588.
SPECIMEN PREPARATION
GRID
SUPPORT
3.05 mm
400 divisons/inch
1 division/66 microns
Copper or other metal
Manufactured “holey carbon grid”
Holes typically 2 microns diameter
2spi.com/catalog
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NEGATIVE STAIN VS. VITREOUS ICE
Specimen in Stain
uranyl acetate
• High contrast image
• No special temperature control
• Essentially no radiation
damage
• Particle distorted
• Image = stain “shell” around
the particle
• Low resolution method: 20-15 Å
• Great choice for initial sample
screening
Cryogenic Specimen
vitreous ice layer
• Low contrast image
• Sample maintained at cryogenic
temperature (85 K)
• High radiation damage
• Particle undistorted
• Image is of the actual particle
• Higher resolution obtained: 15-4 Å
• Best choice for reconstruction
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IMAGING--TEM MICROSCOPE
Sample
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Image: cryoem.berkeley.edu/~nieder
Film or CCD camera
CRYO EM EXPERIMENT
CHARACTERISTICS
Images must be taken with low electron doses to
prevent radiation damage
 3D maps of single particle, filament, and 2D crystal
maps are generated by averaging over thousands of
individual particles
 3D maps of unique structures are generated by
“tomography”.

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Image: http://ami.scripps.edu
DATA COLLECTION AND INITIAL
IMAGE PROCESSING STEPS

Collect image set (20-100 images, vary focus)

Pick Particles (4000-100,000)

Perform contrast-transfer-function (CTF)
correction for particles from each image

Center, align, classify particles
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PARTICLE SELECTION
Raw image
Auto-Select
particles
Particle Composite
For 1 raw image
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CONTRAST TRANSFER FUNCTION
(CTF)
(A) Wedge object
(B) image of wedge object taken slightly out of focus
shows inversions of contrast for increasing spatial frequencies
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Downing & Glaeser, Ultramicroscopy 108: 921-928 (2008).
CTF CORRECTION
Fourier Transform
Of an EM image
Phase inversion
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RECOVERING 3D FROM 2D
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figure from Joachim Frank
RECONSTRUCTION CYCLE
Preliminary 3D Model
Make Projections
Particle Stack
Match Particles
To Projections
Create Class
Averages
Update 3D Model
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RECONSTRUCTION CYCLE
Preliminary 3D Model
Make Projections
Particle Stack
Match Particles
To Projections
Create Class
Averages
Update 3D Model
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EXAMPLE RECONSTRUCTION
Particles (~17000 total)
First refinement cycle
projections
averages
Final refinement cycle
projections
averages
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Brian Hudson, Rutgers
EULER DISTRIBUTION OF CLASSES
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FOURIER SHELL CORRELATION—MAP RESOLUTION
Divide particles into two sets, calculate 2 independent reconstructions
“FSC” measures agreement of the 2 maps as a function of resolution
Position where FSC is 0.5 typically reported as the map resolution
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MAP QUALITY
Common reconstruction resolutions: 8-15 Å
 Rare: 4-5 Å
 Theoretical limit: < 2 Å

Sources of degradation of map quality:
 Specimen
 Imaging
 Data processing
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MAP QUALITY—RIBOSOME
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figure from Joachim Frank
STRUCTURE ANALYSIS
Methods to interpret EM map volumes:
 SEGMENTATION – identifying contiguous parts
of the map representing individual components or
chains.
 FITTING – placement of atomic coordinate
models from other experimental methods.
 REFINEMENT – adjusting atomic model to
improve fit to the map.
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SEGMENTATION
(a) herpes simplex capsid heterotrimer
(b) Epsilon 15 bacteriophage
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Images from Wah Chiu
FITTING
Rossmann, M. G. et al 2004. Curr Opin Struct Biol 14:171-80.
Kostyuchenko, V. A. et al 2005 Nat Struct Mol Biol 12:810-3.
EMDB: EMD-1048
PDB: 1pdf, 1pdi, 1pdl
1pdm, 1pdp, 2fl8
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COMPOSITE MODEL
T. thermophilus RNAP holoenzyme
Mukhopadhyay et al. (2008) 3dxj
CAP + αCTD + DNA
Benoff et al. (2002) 1lb2
αCTD + σ70 region 4 + DNA
Lara-Gonzalez et al, ms in prep
DNA positions -23 to +20
Lawson et al. (2004) model
E. coli αNTD dimer
Zhang & Darst (1998) 1bdf
E. coli σ70 regions 1.2 to 2
Malhotra et al. (1996) 1sig
E. coli β’GNCD
Chlenov et al. (2005) 2auk
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3iyd
Hudson et al (2009)
3D VISUALIZATION MAP+MODEL
http://www.emdatabank.org/emviz/5127-3iyd-v3a.html
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REFERENCES
Frank, J. 2006. Three-dimensional electron microscopy of
macromolecular assemblies : visualization of biological
molecules in their native state. 2nd ed. New York : Oxford
University Press.
Chiu, W., M. L. Baker, and S. C. Almo. 2006. Structural
biology of cellular machines. Trends Cell Biol 16:144-50.
Nickell, S., C. Kofler, A. P. Leis, and W. Baumeister. 2006.
A visual approach to proteomics. Nat Rev Mol Cell Biol
7:225-30.
Taylor, K. A. and Glaeser, R.M. 2008. Retrospective on the
early development of cryoelectron microscopy of
macromolecules and a prospective on opportunities for the
future. J. Struct. Biol. 163:214-223.
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RESOURCES

Structural Biology EM Research Resource Centers






NCMI: http://ncmi.bcm.tmc.edu/ncmi/
NRAMM: http://nramm.scripps.edu/
NCMIR: http://www-ncmir.ucsd.edu/
RVBC: http://www.wadsworth.org/rvbc/
Bio3D: http://bio3d.colorado.edu/
EM software tools:

http://en.wikipedia.org/wiki/
Software_tools_for_molecular_microscopy
EMDB: emdatabank.org
 Icosahedral Virus database: viperdb.scripps.edu

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MOVIES BASED ON CRYOEM DATA


Microtubules: http://cryoem.berkeley.edu/animations.shtml
T4 virus: www.nsf.gov/news/news_summ.jsp?
cntn_id=100420&org=MCB&from=news


Ribosome: www.wadsworth.org/databank/electron
Actomyosin/kinesin: www.scripps.edu/cb/milligan/index.html
T4 virus
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