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

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Presentation on theme: "Basic Electron Microscopy Arthur Rowe The Knowledge Base at a Simple Level."— Presentation transcript:

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

2 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 > 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

4 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

5 ways 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

6 negative staining Electron dense negative stain particles

7 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: anioniccationic phosphotungstateuranyl actetate/formate molybdate pH ~ 4)

8 metal shadowing - 1-directional

9 Contrast usually inverted to give dark shadows > resolution 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

10 metal shadowing - rotary

11 Contrast usually inverted to give dark shadows > resolution 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

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

13 particle in vitrified ice: low contrast average of large numbers (thousands +) of very low contrast particles enables a structure to be determined

14 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

15 particle in vitrified ice: the ribosome

16 particle in vitrified ice: phage T4 & rotavirus

17 case study : GroEL-GroES important chaperonins hollow structure appear to require ATP (hydrolysis ?) for activity

18 particle in vitrified ice: low contrast the chaperonin protein GroEL visualised in vitrified ice (Helen Saibil & co-workers)

19 GroEL GroEL + ATP GroEL+GroES +ATP

20 DLS as a probe for conformational change in GroEL/ES

21 GroEL GroEL + ATP GroEL+GroES +ATP

22

23 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

24 electron micrographs of pores in membranes caused by pneumolysin RBC / negative staining membrane fragment metal shadowed

25 Pneumolysi n Homology model based upon the known crystallographic structure of Perfringolysin

26 Pneumolysin - homology model ± domain 3, fitted to cryo reconstruction

27 Pneumolysin - EM by microcrystallite decoration (MCD) reveals orientation of domains

28 Pneumolysin - monomers identified within the oligomeric form (i.e. the pore form)

29 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

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

31 Each S1 unit has a compact region, & a lever arm connected via a hinge to the main extended tail

32 Myosin S1 imaged by Microcrystallite Decoration (no nucleotide present)

33 -ADP+ADP Effect of nucleotide (ADP) on the conformation of myosin S1 as seen by MCD electron microscopy

34 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: S15 nm S110 nm S25 nm

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

36 triple labelling of 3 epitopes on hepagene

37 Basic Electron Microscopy Arthur Rowe End


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