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

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

Curr Protoc Protein Sci. 2005 Dec;Chapter 17:Unit 17.2

Plunge Freezing

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

Curr Protoc Protein Sci. 2005 Dec;Chapter 17:Unit 17.2

Physiology 21: 13-18, 2006

Cryo-EM + X-ray

Combining methods for pseudo-atomic models

Sampling of Icosahedral Virus Cryo-EM Reconstructions (1999)

EMDB Stats

http://www.ebi.ac.uk/pdbe/emdb/statistics.html

Current Structures

Highest Resolution in EMDB: Helical – 2.8 Icosahedral – 3.1

EMBO J. 2011 Jan 19;30(2):408-16. EMBO J. 2011 Jan 19;30(2):408-16.

4.3Å

26Å

What did we learn withhigher resolution?

Significance

The value of the results produced by 3D reconstruction ofviruses from cryo-EM must be considered in terms of its contributionto 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 between capsomers

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