Basic Electron Microscopy

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Basic Electron Microscopy

• Arthur Rowe

The Knowledge Base at a Simple Level
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Introduction

• These 3 presentations cover the fundamental
  theory of electron microscopy
• In presentation 3 we cover
• requirements for imaging macromolecules
• aids such as gold-labelled antibodies
• the negative staining method
• the metal-shadowing method
• Including high-resolution modifications
• vitritied ice technology
• examples of each type of method

3
requirements for imaging macromolecules

• sufficient CONTRAST must be attainable, but
• gt bio-molecules are made up of low A.N. atoms
• gt are of small dimensions (4 nm)
• gt hence contrast must usually be added
• sufficient STABILITY in the beam is needed
• gt to enable an image to be recorded
• gt low dose random imaging mandatory for any
• high resolution work

4
ways of imaging macromolecules

• ADDING CONTRAST (with heavy metals)
• gt negative contrast
• computer analysis
• immunogold labels
• gt metal shadowing
• computer enhancement
• USING INTRINSIC CONTRAST
• gt particles in thin film of vitrified ice
• computer acquisition processing

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ways of imaging macromolecules

• using immunogold labels to localise epitopes
• gt widely used in cell biology
• gt beginning to be of importance for
  macromolecules

Au sphere Mab epitope
macromolecule
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negative staining
particles
Electron dense negative stain
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negative staining

• requires minimal interaction between particle
  stain
• to avoid binding, heavy metal ion should be of
  same charge /- as the particle
• positive staining usually destructive of
  bio-particles
• biological material usually -ve charge at
  neutral pH
• widely used negative contrast media include
• anionic cationic
• phosphotungstate uranyl actetate/formate
• molybdate (ammonium) (_at_ pH 4)

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metal shadowing - 1-directional
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metal shadowing - 1-directional

• Contrast usually inverted to give dark shadows
• gt resolution 2 - 3 nm - single 2-fold a-helix
  detectable
• - historic use for surface detail
• - now replaced by SEM
• gt detail on shadow side of the particle can be
  lost
• gt apparent shape can be distorted
• gt problems with orientation of elongated
  specimens
• - detail can be lost when direction of
• shadowing same as that of feature
• gt very limited modern use for macromolecular work

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metal shadowing - rotary
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metal shadowing - rotary

• Contrast usually inverted to give dark shadows
• gt resolution 2 - 3 nm - single DNA strand
  detectable
• - historic use for molecular biology
• (e.g. heteroduplex mapping)
• gt good preservation of shape, but enlargement of
• apparent dimensions
• gt in very recent modification (MCD -
  microcrystallite
• decoration), resolution 1.1 nm

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particle in vitrified icelow contrast
particle
particles examined at v. low temperature, frozen
in a thin layer of vitrified (structureless) ice
- i.e. no contrast added
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particle in vitrified icelow contrast
average of large numbers (thousands ) of very
low contrast particles enables a structure to be
determined
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particle in vitrified icelow contrast

• average of large numbers (thousands ) of very
  low contrast particles enables a structure to be
  determined
• resolution may be typically 1 nm or better
• this is enough to define the outline (or
  envelope) of a large structure
• detailed high resolution data give us models for
  domains (or sub-domains) which can be fitted
  into the envelope
• ultimate resolution of the method 0.2 nm,
  rivalling XRC/NMR

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particle in vitrified icethe ribosome
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particle in vitrified icephage T4 rotavirus
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case study GroEL-GroES

• important chaperonins
• hollow structure
• appear to require ATP (hydrolysis ?) for
  activity

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particle in vitrified icelow contrast
the chaperonin protein GroEL visualised in
vitrified ice (Helen Saibil co-workers)
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GroEL GroEL ATP GroELGroES
ATP
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GroEL GroEL ATP GroELGroES
ATP
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case study pneumolysin
53 kD protein, toxin secreted from
Pneumococcus pneumoniae among other effects,
damages membrane by forming pores major
causative agent of clinical symptoms in
pneumonia
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electron micrographs of pores in membranes
caused by pneumolysin
RBC / negative staining
membrane fragment metal shadowed
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Pneumolysin Homology model based upon the known
crystallographic structure of Perfringolysin
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Pneumolysin - homology model domain 3, fitted
to cryo reconstruction
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Pneumolysin - EM by microcrystallite decoration
(MCD) reveals orientation of domains
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Pneumolysin - monomers identified within the
oligomeric form (i.e. the pore form)
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case study myosin S1
motor domain of the skeletal muscle protein
myosin 2 S1s / myosin, mass c. 120 kD
cross-bridge between myosin and actin
filaments, thought to be source of force
generation
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myosin is a 2-stranded coiled-coil protein, with
2 globular (S1) heads
S1 unit
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Each S1 unit has a compact region, a lever
arm connected via a hinge to the main extended
tail
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Myosin S1 imaged by Microcrystallite Decoration
(no nucleotide present)
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Effect of nucleotide (ADP) on the conformation of
myosin S1 as seen by MCD electron microscopy
-ADP
ADP
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case study epitope localisation in an
engineered vaccine
a new vaccine for Hepatitis B contains 3
antigens, S, S1 S2, with epitopes on each but
does every particle of hepagene contain all
3 of these epitopes ? Mabs against S, S1 S2
have been made conjugated with
gold S 15 nm S1 10 nm S2 5 nm
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immunolabelling of one epitope (S1) in hepagene
using 10 nm-Au labelled Mab
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triple labelling of 3 epitopes on hepagene
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Basic Electron Microscopy

• Arthur Rowe

End