partie EM cours EMBO

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• Publié par: Uste
• Nombre de lectures: 18
• Langue: English

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EMBO Practical Course 2004: EM Workshop 1
EMBO Practical Course:
Structural Characterization of Macromolecular Complexes:
Modern Techniques and Strategies
Grenoble Outstation, May 3-8, 2004
Workshop on Electron Microscopy, Analysis and Fitting
1. Electron microscopy (negative staining of adenovirus particles)
Electron microscopy (EM) can be used to obtain qualitative (purity and homogeneity) and
quantitative (concentration, size and oligomeric state) information. It is a fast method (15
min.) and gives direct imaging of the protein sample (see Figure below). An electron
microscope will be used for demonstrating negative-stain preparation, imaging and data
collection. Students will have the opportunity to discover the ease of this method and the
important information it can provide. [1]
Details. A 3µl sample of virus capsids at a concentration of ~0.1 mg/ml is adsorbed to a
carbon-mica interface. The carbon film with adsorbed protein is floated onto a solution of the
negative stain, which contains heavy metal ions – a 1% solution of ammonium molybdate
was used in the image below. The film is picked up by a copper EM grid and then air-dried.
The sample is embedded in the stain, which protects it from the EM vacuum as well as
providing high contrast due to the strong scattering power of the metal ions. The samples will
be imaged in a JEOL 1200 EXII electron microscope at a magnification of ~30000.
Conditions of low electron dose can be used to preserve the structure as much as possible.

Electron microscopy of adenovirus in negative stain (top) and by cryoEM
(bottom). Although more difficult, cryoEM avoids structural disturbances from
staining and dehydration and is the method of choice for high resolution EM
studies.
EMBO Practical Course 2004: EM Workshop 2
2. Data analysis (cryoEM images of adenovirus sample)
Three-dimensional analysis of particulate specimens will be demonstrated briefly, using
adenovius particles imaged by cryo-electron microscopy. A dataset of scanned images will be
used to show the more interactive steps including particle selection, calculation of power
spectra, estimation of image distortions (eg, the contrast transfer function of the microscope)
and their correction. The results of an analysis will be presented along with some guidelines
for interpreting the density and the quality of the density map. [2, 3]


Surface view of a 3D reconstruction of the adenovirus capsid at 25 Å resolution.
Icosahedral symmetry is evident in the positioning of pentavalent protrusions at
vertices (eg, left and right of mid-plane), each with five trimeric neighbours.
Trimers are in hexavalent positions, according to a geometry of triangulation
number T=25. (For a review of icosahedral geometry employed by viruses, see
[4])



EMBO Practical Course 2004: EM Workshop 3
3. Modelling
Fitting atomic models of subunits into a 10-15Å resolution density map from cryo-EM is an
important technique for studying protein complexes. Docking with the SITUS software will
be shown on the adenovirus EM envelope. The structure of the two major components of the
capsid are know and will be fitted into the EM envelope with assistance from the author,
Prof. Willy Wriggers. [3, 5, 6]


Fit of the hexon into the EM enveloppe


References
1. Valentine RC, Shapiro BM & Stadtman ER (1968) Regulation of glutamine synthetase.
XII. Electron microscopy of the enzyme from Escherichia Coli. Biochemistry 7, 2143-
2152.
2. Conway JF & Steven AC (1999) Methods for reconstructing density maps of “single”
particles from cryoelectron micrographs to subnanometer resolution. J Struct Biol 128,
106-118.
3. Conway JF, Wikoff WR, Cheng N, Duda RL, Hendrix RW, Johnson JE & Steven AC
(2001) Virus maturation involving large subunit rotations and local refolding. Science.
292, 744-748.
4. Johnson JE & Speir JA (1997) Quasi-equivalent viruses: a paradigm for protein
assemblies. J Mol Biol 269, 665-675.
5. Wriggers W, Milligan RA & McCammon JA (1999) Situs: A package for docking crystal
structures into low-resolution maps from electron microscopy. J Struct Biol 125, 185-195.
6. Wriggers W & Birmanns S. (2001). Using situs for flexible and rigid-body fitting of
multiresolution single-molecule data. J Struct Biol 133, 193-202.