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Basic Electron Microscopy. Arthur Rowe. The Knowledge Base at a Simple Level. 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

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Basic electron microscopy l.jpg

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


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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


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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


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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


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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) (@ pH ~ 4)



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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



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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


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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


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particle in vitrified ice:low contrast

average of large numbers (thousands +) of very low contrast particles enables a structure to be determined


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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



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particle in vitrified ice:phage 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 ice:low contrast

the chaperonin protein GroEL visualised in vitrified ice

(Helen Saibil & co-workers)





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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 clinicalsymptoms 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 reveals orientation of domains

- monomers

identified within the oligomeric form (i.e. the pore form)


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case study : myosin S1 reveals orientation of domains

• 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



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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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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 as seen by MCD electron microscopy

• 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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Basic Electron Microscopy nm-Au labelled Mab

Arthur Rowe

End