intro to cryo-em and icosahedral symmetry lab meeting 6-16-2011
TRANSCRIPT
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Intro to Cryo-EM and Icosahedral Symmetry
Lab Meeting
6-16-2011
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Why EM?
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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.
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Why Cryo-EM?
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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
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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
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Cryo-EM + X-ray
Combining methods for pseudo-atomic models
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Sampling of Icosahedral Virus Cryo-EM Reconstructions (1999)
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EMDB Stats
http://www.ebi.ac.uk/pdbe/emdb/statistics.html
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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?
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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.
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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.
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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.
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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
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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
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Example
http://www.cgl.ucsf.edu/chimera/experimental/flatten_icosahedron/flaticos.html
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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.
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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
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Examples
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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.
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Examples
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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