1. Intro to Cryo-EM and Icosahedral Symmetry
Lab Meeting

3. Why EM?

4. Adapted from review by Subramaniam in Current Opinion In Microbiology (2005),
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.

5. Why Cryo-EM?

6. Negative Stain Advantages Disadvantages high contrast
Curr Protoc Protein Sci 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

7. Plunge Freezing Advantages preservation of native structure
Curr Protoc Protein Sci 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

8. Combining methods for pseudo-atomic models
Cryo-EM X-ray

9. Sampling of Icosahedral Virus Cryo-EM Reconstructions (1999)

10. EMDB Stats

11. Current Structures 26Å What did we learn with 4.3Å higher resolution?
EMBO J. 2011 Jan 19;30(2):
EMBO J. 2011 Jan 19;30(2):
What did we learn with
higher resolution?
4.3Å
Highest Resolution in EMDB: Helical – 2.8
Icosahedral – 3.1

12. Significance The value of the results produced by 3D reconstruction of
viruses from cryo-EM must be considered in terms of its contribution
to our understanding of viral structural biology.

13. 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.

14. 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.

15. 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 between capsomers

16. 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.

17. Example

18. 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.

19. 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

20. Examples

21. 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.

22. Examples

23. Useful Websites http://www.virology.wisc.edu/virusworld/tri_number.php