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

Basic Electron MicroscopyBasic Electron Microscopy

Arthur Rowe

The Knowledge Base at a Simple Level

Introduction 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

requirements for imaging requirements for imaging macromoleculesmacromolecules

• 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 macromoleculesways 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 macromoleculesways of imaging macromolecules• using immunogold labels to localise epitopes

> widely used in cell biology

> beginning to be of importance for macromolecules

macromolecule

Au sphere

Mab

epitope

negative stainingnegative staining

Electron dense negative stain

particles

negative stainingnegative 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-directionalmetal shadowing - 1-directional

metal shadowing - 1-directionalmetal 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 - rotarymetal shadowing - rotary

metal shadowing - rotarymetal 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

particle in vitrified ice:particle in vitrified ice:low contrastlow contrast

particles examined at v. low temperature, frozen in a thin layer of vitrified (structureless) ice - i.e. no contrast added

particle in vitrified ice:particle in vitrified ice:low contrastlow contrast

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

particle in vitrified ice:particle in vitrified ice:low contrastlow 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:particle in vitrified ice:the ribosomethe ribosome

particle in vitrified ice:particle in vitrified ice:phage T4 & rotavirusphage T4 & rotavirus

case study : GroEL-GroEScase study : GroEL-GroES

• important chaperonins

• hollow structure

• appear to require ATP (hydrolysis ?) for activity

particle in vitrified ice:particle in vitrified ice:low contrastlow contrast

the chaperonin protein GroEL visualised in vitrified ice

(Helen Saibil & co-workers)

GroEL GroEL + ATP GroEL+GroES +ATP

3.91

5.40

8.54

2.55

8.49

2.60

8.48

2.51

9.15

2.41

10.19

2.20

2

3

4

5

6

7

8

9

10

11

ES EL ELES ELES+ ATP-gamma-s

ELES+0.5mMATP

ELES+2mMATP

Rh(nm)D20w (x10-7)

DLS as a probe for conformational change in GroEL/ES

GroEL GroEL + ATP GroEL+GroES +ATP

case study : pneumolysincase 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 electron micrographs of pores in

membranes caused by pneumolysinmembranes caused by pneumolysin

RBC / negative staining membrane fragment metal shadowed

Pneumolysin

Homology model based upon the

known crystallographic

structure of

Perfringolysin

Pneumolysin - homology model ± 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 S1case 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

S1 unit

myosin is a 2-stranded coiled-coil protein, with 2 globular (S1) ‘heads’

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)

-ADP +ADP

Effect of nucleotide (ADP) on the conformation of myosin S1 as seen by

MCD electron microscopy

case study : epitope localisation case study : epitope localisation in an engineered vaccinein 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:

SS 15 nm15 nm

S1S1 10 nm10 nm

S2S2 5 nm5 nm

immunolabelling of one epitope (S1) in hepagene using 10 nm-Au labelled Mab

triple labelling of 3 epitopes on hepagene

Basic Electron MicroscopyBasic Electron Microscopy

Arthur Rowe

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