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Basic Electron Microscopy

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Basic Electron Microscopy
Arthur Rowe
The Knowledge Base at a Simple Level
Introduction
пЃџ These
3 presentations cover the
fundamental theory of electron microscopy
пЃџ In presentation #3 we cover:
–
requirements for imaging macromolecules
_
–
–
the negative staining method
the metal-shadowing method
_
–
–
aids such as gold-labelled antibodies
Including high-resolution modifications
vitritied ice technology
examples of each type of method
requirements for imaging
macromolecules
• sufficient CONTRAST must be attainable, but
> bio-molecules are made up of low A.N. atoms
> & are of small dimensions (4+ nm)
> hence contrast must usually be added
• sufficient STABILITY in the beam is needed
> to enable an image to be recorded
> low dose �random’ imaging mandatory for any
high resolution work
ways of imaging macromolecules
• ADDING CONTRAST (with heavy metals)
> negative contrast
+ computer analysis
+ immunogold labels
> metal shadowing
+ computer enhancement
• USING INTRINSIC CONTRAST
> particles in thin film of vitrified ice
+ computer acquisition & processing
ways of imaging macromolecules
• using immunogold labels to localise epitopes
> widely used in cell biology
> beginning to be of importance for macromolecules
Au sphere
Mab
epitope
macromolecule
negative staining
particles
Electron dense
negative stain
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)
(@ pH ~ 4)
metal shadowing - 1-directional
metal shadowing - 1-directional
• Contrast usually inverted to give dark shadows
> resolution 2 - 3 nm - single 2-fold a-helix detectable
- historic use for surface detail
- now replaced by SEM
> detail on �shadow’ side of the particle can be lost
> apparent �shape’ can be distorted
> problems with orientation of elongated specimens
- detail can be lost when direction of
shadowing same as that of feature
> very limited modern use for macromolecular work
metal shadowing - rotary
metal shadowing - rotary
• Contrast usually inverted to give dark shadows
> resolution 2 - 3 nm - single DNA strand detectable
- historic use for �molecular biology’
(e.g. heteroduplex mapping)
> good preservation of shape, but enlargement of
apparent dimensions
> in very recent modification (MCD - microcrystallite
decoration), resolution ~1.1 nm
particle in vitrified ice:
low contrast
particle
particles examined at v. low temperature, frozen in a thin layer
of vitrified (structureless) ice - i.e. no contrast added
particle in vitrified ice:
low contrast
average of large numbers (thousands +) of very low contrast
particles enables a structure to be determined
particle in vitrified ice:
low 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
particle in vitrified ice:
the ribosome
particle in vitrified ice:
phage T4 & rotavirus
case study : GroEL-GroES
• important chaperonins
• hollow structure
• appear to require ATP (hydrolysis ?) for
activity
particle in vitrified ice:
low contrast
the chaperonin protein GroEL visualised in vitrified ice
(Helen Saibil & co-workers)
GroEL
GroEL + ATP
GroEL+GroES
+ATP
DLS as a probe for conformational change in GroEL/ES
10.19
11
9.15
10
8.54
8.49
8.48
9
8
Rh(nm)
7
D20w (x10-7)
6
5
5.40
3.91
4
2.60
2.55
2.51
2.41
2.20
3
2
ES
EL
ELES
ELES+ ATPgamma-s
ELES+
0.5mMATP
ELES+
2mMATP
GroEL
GroEL + ATP
GroEL+GroES
+ATP
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
electron micrographs of pores in
membranes caused by pneumolysin
RBC / negative staining
membrane fragment metal shadowed
Pneumolysi
n
Homology model
based upon the
known
crystallographic
structure of
Perfringolysin
Pneumolysin - homology model В± domain 3,
fitted to cryo reconstruction
Pneumolysin - EM by microcrystallite decoration
(MCD) reveals orientation of domains
Pneumolysin
- monomers
identified
within the
oligomeric
form (i.e. the
pore form)
case study : myosin S1
• motor domain of the skeletal muscle
protein myosin
• 2 S1’s / myosin, mass c. 120 kD
• �cross-bridge’ between myosin and
actin filaments, thought to be
source of force generation
myosin is a 2-stranded coiled-coil protein,
with 2 globular (S1) �heads’
S1 unit
Each S1 unit has a compact region, & a �lever arm’
connected via a �hinge’ to the main extended �tail’
Myosin S1 imaged by Microcrystallite
Decoration (no nucleotide present)
Effect of nucleotide (ADP) on the conformation of
myosin S1 as seen by MCD electron microscopy
-ADP
+ADP
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
immunolabelling of one epitope (S1) in hepagene
using 10 nm-Au labelled Mab
triple labelling of 3 epitopes on hepagene
Basic Electron Microscopy
Arthur Rowe
End
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