Electron Microscopy

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Cryo-Electron Microscopy Methods February 4,
2005 Phoebe L. Stewart Molecular Physiology
Biophysics phoebe.stewart_at_vanderbilt.edu
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Suitable Samples for CryoEM
Saibil, Acta Cryst. 2000, D561215
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Single Particles - Suitable Samples (200 kDa
400 MDa)
Asymmetric monomeric proteins e.g. DNA-PKcs,
470 kDa
Protein/RNA or DNA complexes e.g. Ribosome, 2
MDa
Icosahedral viruses 60-fold symmetry e.g.
Adenovirus, 150 MDa
Detergent solubilized membrane proteins
e.g. Voltage-sensitive sodium channel 300 kDa,
Sato et al., Nature 2001
e.g. Platelet integrin aIIbb3 230 kDa, Adair
Yeager, PNAS 2002
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Single Particle Method - Overview
1. Cryo-Plunge Samples
2. Collect Micrographs
3. Digitally Select Particle Images
4. 3D Image Processing
5. Structural Analysis Modeling
GOAL ? Use structural information to understand
biological function
Average 100s or 1000s of particle images
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1. Cryo-Plunge Samples EM Sample Grids

• For traditional electron microscopy, a copper
  mesh grid is covered with a thin carbon film.

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• For cryo-EM, a copper mesh grid is covered with a
  holey carbon film (A). Images are collected of
  the frozen sample suspended in holes of the
  carbon film (B). Any particles that are on the
  carbon support are not used in the image
  processing because they have a higher background
  signal.

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Cryo-Plunging Device

• The biological sample is applied to the grid,
  blotted to leave a thin film of water, and then
  plunge-frozen in ethane slush chilled by liquid
  nitrogen.
• Cryo sample preparation methods were developed in
  the mid 1980s

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Cryo Plunging and Grids
Home built Plunger
Vitrobot Reproducibility
Quantifoil Grids Automation of Data
Acquisition LEGINON System Scripps
Homemade Holey film
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Three Forms of Ice

• Plunge freezing of a thin (1,000Å) layer of
  water into a cryogen produces vitrified ice, or
  water in a glass-like state.
• The diffraction pattern of vitrified ice shows no
  regular diffraction spacing, indicating a
  non-crystalline structure.
• Hexagonal ice is the normal crystalline form of
  ice.
• Cubic ice is formed when vitrified ice warms up
  above approx. -130C.
• Cubic and hexagonal ice both have a greater
  volume than liquid water - expansion occurs
  during non-cryogenic freezing.
• This expansion would distort the 3D structure of
  the biological sample.

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2. Collect Cryo-Micrographs
Cryo Sample Holder

• The frozen sample grid is normally kept at
  liquid nitrogen temperature (approximately
  -185C) while in the vacuum of the microscope by
  a cryo-holder.
• Liquid helium microscopes allow the sample grid
  to be kept even colder (12 K, -261C) in the
  microscope.

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Transmission Electron Microscope (TEM)

• A high energy electron beam is focused by
  magnetic lenses, the column is under ultra-high
  vacuum, the sample must be dry or frozen.

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Electro-Magnetic Spectrum

• The electron wavelength is much shorter than 1
  Å (10-10 m) and does not limit the resolution in
  TEM.
• Note, electrons are particles with a dual wave
  nature, rather than part of the traditional
  electromagnetic spectrum.

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TEM Image Collection

• A digital cryo-micrograph of adenovirus (shown
  artificially colored to enhance the image
  contrast)

• The images are collected by
• exposing and developing a piece of photographic
  film (the negative has to be scanned if you want
  to do image processing)
• or by digitally collecting the image on a CCD
  (charge coupled device) camera

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Radiation Damage

• A low dose of electrons must be used to avoid
  radiation damage

• A cryo-electron micrograph of adenovirus showing
  radiation damage. The virus particles have been
  destroyed by the electron beam.

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Microscope and Camera

• Both the type of microscope and camera can limit
  the resolution

120kV non-FEG Nitrogen
300kV FEG Nitrogen Helium High Coherence
4k x 4k camera 0.5 Å pixel High Resolution
1k x 1k camera 4 Å pixel

• Expect 15-20 Å resolution

• Expect 1-5 Å resolution

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3. Digitally Select Particle Images

• Individual particle images must be selected from
  digital cryo-electron micrographs. This has been
  done interactively (non-automatically) in the
  past. Software is being developed for automatic
  particle selection.

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4. Three-Dimensional Image Processing

• Each particle image represents a 2D projection of
  the 3D object

3D object (a duck)
2D projections in different views

• The difficult step in 3D image processing is to
  determine the orientational angles (Euler angles)
  for each projection image

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How many projection images are needed to generate
a 3D reconstruction?

• A single projection image is plainly insufficient
  to infer the structure of an object.
• (Originally from The New Yorker Magazine, 1991)

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If the particle has no symmetry, projection
images are needed for essentially all possible
views

• This Euler sphere representation (which is like a
  flat map of the globe) shows each projection
  image in a cryoEM data set as a dot
• The entire Euler sphere should be spanned for an
  asymmetric particle.

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If the particle has symmetry, fewer projection
images are needed

• For a highly symmetric object, such as an
  icosahedral (60-fold symmetric) virus, projection
  images only need to be collected within the
  asymmetric unit (1/60th of the surface).
• The number of projection images also affect the
  resolution that is attainable

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3D Image Processing
Particle images (with determined Euler angles)
3D Reconstruction
Reprojections (should match particle images)
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Resolution Assessment

• FOURIER SHELL CORRELATION (FSC) METHOD - The data
  set is split into two halves and two independent
  half reconstructions are calculated
• A correlation coefficient is then calculated
  between the two half reconstructions and plotted
  as a function of resolution
• The resolution estimate is where the FSC
  correlation falls to 0.5

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5. Structural Analysis Modeling
Structural Analysis at 20 Å Resolution

• If 20 Å resolution is reached, approaches such
  as peptide tagging, antibody labeling, and
  fitting the cryoEM density with atomic structures
  of components all help to interpret the cryoEM
  density.

Hepatitis B capsid with an 8 residue peptide tag
(red) added to the N-terminus of the recombinant
capsid protein. Conway et al., PNAS 1998 9514622
CryoEM reconstruction of adenovirus (just one
capsid protein is shown in yellow) complexed with
a monoclonal antibody (red) analyzed with the
atomic structure of the major capsid protein
(magenta, green, blue). Varghese et al., J.
Virol. 2004 7812320
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Structural Analysis at 10 Å Resolution

• If 10 Å resolution is reached, it is possible to
  resolve alpha helices in the cryoEM density
• It is also possible to determine if a predicted
  homology model for a protein domain agrees with
  the shape of the cryoEM density

Jiang et al., 2001, J. Mol. Biol. Helixhunter
Rice Dwarf Virus Zhou et al. 2001, Nat. Struct.
Biol.
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Structural Analysis at 1 Å Resolution

• If 1 to 4.5 Å resolution is reached, it should
  be possible to determine atomic resolution
  structures (similar to x-ray crystallography)
• The best published resolution by cryoEM single
  particle reconstruction so far is 7 Å
• Other cryoEM methods (2D crystals and helical
  filaments) have reached 3 to 4.5 Å resolution, so
  similar resolutions should be possible by single
  particle reconstruction
• It is predicted that 1 million particle images
  will be needed to reach 3 Å resolution
• The number of particle images may be divided by
  the symmetry. So for an icosahedral virus, 1
  million/60 16,000

X-ray crystallography density (red) and atomic
model (yellow) Terwilliger, LANL,
website www.solve.lanl.gov/index.html
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Summary CryoEM Methods

• No crystals are needed
• Cryo freezing preserves the 3D structure of the
  sample. (Traditional sample staining usually
  produces structural distortions)
• The electron dose must be kept low to avoid
  damaging the frozen sample
• There is low image contrast in cryo-electron
  micrographs
• Many particle images (projection images) must be
  averaged to generate a 3D reconstruction
• The difficult part is determining the Euler
  (orientational) angles for each projection image
• Biological complexes in the range of 300kDa to
  400 MDa may be imaged (100 - 1,500 Å in diameter)
• Typical resolutions after 3D reconstruction are
  7-25 Å
• 3-4.5 Å resolution may be feasible with high-end
  cryo-electron microscopes and by averaging of 1
  million particle images

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CryoEM - Applications
On Monday (2/7) we will discuss two cryoEM
papers Z. Hong Zhou et al., Electron
cryomicroscopy and bioinformatics suggest protein
fold models for rice dwarf virus. (2001) Nat.
Struct. Biol. 8868 Brian D. Adair and Mark
Yeager, Three-dimensional model of the human
platelet integrin IIb3 based on electron
cryomicroscopy and x-ray crystallography. (2002)
PNAS 9914059