Intro to Cryo-EM and
Icosahedral Symmetry
Lab Meeting
6-16-2011
Why EM?
Adapted from review by Subramaniam in Current Opinion In Microbiology (2005),
http://electron.nci.nih.gov/html/home.html
Developments in electron microscopy have generated a renaissance in biological imaging,
allowing researchers to visualize 3D structures of biological entities at the molecular scale including viruses, protein complexes and individual proteins.
Why Cryo-EM?
Negative Stain
Curr Protoc Protein Sci. 2005 Dec;Chapter 17:Unit 17.2
Advantages
• high contrast
• good signal-to-noise ratio
• quick and easy to learn
• simple to apply
• resistant to radiation damage
• good for small objects (proteins)
Disadvantages
• distortion due to dehydration on support
• artifacts due to staining pattern
• high background from surrounding stain
• distortions due to ionic strength and pH
• limited resolution
• surface contour only
Plunge Freezing
Curr Protoc Protein Sci. 2005 Dec;Chapter 17:Unit 17.2
Physiology 21: 13-18, 2006
Advantages
• preservation of native structure
• always in solution and hydrated
• high resolution
• internal details revealed
Disadvantages
• sophisticated equipment
• learning curve
• low signal-to-noise ratio
• usually requires >200kD
Cryo-EM suite and trained microscopist operational in Hershey by Feb 2012
Combining methods for pseudo-atomic models
Cryo-EM
+
X-ray
Sampling of Icosahedral Virus Cryo-EM Reconstructions
(1999)
EMDB Stats
http://www.ebi.ac.uk/pdbe/emdb/statistics.html
Current Structures
26Å
EMBO J. 2011 Jan 19;30(2):408-16.
4.3Å
EMBO J. 2011 Jan 19;30(2):408-16.
What did we learn with
higher resolution?
Highest Resolution in EMDB: Helical – 2.8
Icosahedral – 3.1
Significance
The value of the results produced by 3D reconstruction of
viruses from cryo-EM must be considered in terms of its contribut
to our understanding of viral structural biology.
Icosahedral Symmetry
• Since a virus structure is optimized for the
propagation of its genome,it is
advantageous to make a large shell with a
small amount of information devoted to
structural components.
• An icosahedron is an isometric structure
with 12 pentagonal vertices and 20
triangular faces.
Icosahedral Symmetry
• Any icosahedron has a defined set of exact
symmetry elements:
– 6 five-fold axes through the 12 vertices
– 10 three-fold axes through the 20 triangular faces
– 15 two-fold axes through the edges
• This means that the complete structure can be
generated by taking 1/60th, called the
asymmetric unit, and operating on it with the
symmetry elements.
Quasi-equivalence
• Simplest icosahedral structure:
60 identical subunits interact
identically
• Caspar and Klug: if >60
subunits interact to form
closed shell, all subunits
cannot have identical
environments.
• They are quasi-equivalent
because their environments
were similar but not identical
– bonds between subunits in a
capsomer are stronger than bonds
http://www.virology.wisc.edu/virusworld/tri_number.php
Building an Icosahedron
• To make isometric shell, begin with a
flat, hexagonal net.
• To curve the net and generate a closed
structure, convert some of the hexagons
to pentagons.
http://www.virology.wisc.edu/virusworld/tri_number.php
Example
http://www.cgl.ucsf.edu/chimera/experimental/flatten_icosahedron/flaticos.html
Triangulation
Numbers
• The larger icosahedra have hexagons between
the pentagons and can be visualized as replacing
the original triangular faces with larger ones
formed from equilateral triangles.
• The number of triangles replacing the original one
is the triangulation number.
Triangulation
Numbers
• T = h2 + hk + k2
• Original theory of quasi-equivalence: number
of different environments should equal the
triangulation number
– a T=4 virus would have four different subunit
environments and 60T (240) subunits
– 10(T-1) hexamers plus 12 pentamers
– not always strictly maintained
Examples
Handedness
Spherical viruses with T numbers greater than or equal to 7 are skewed.
They are therefore described as either right- (dextro) or left- (laevo) handed.
Examples
Useful Websites
• http://www.virology.wisc.edu/virusworld/tri_number.php
• http://www.nlv.ch/Virologytutorials/Structure.htm
• http://web.uct.ac.za/depts/mmi/stannard/virarch.html
• http://viperdb.scripps.edu/icos_server.php