Basic Electron Microscopy

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 Au sphere Mab epitope macromolecule

6. negative staining particles Electron dense negative stain

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: • anionic cationic • phosphotungstate uranyl actetate/formate • molybdate (ammonium) (@ pH ~ 4)

8. metal shadowing - 1-directional

9. 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

10. metal shadowing - rotary

11. 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

12. 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

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

23. 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

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

25. Pneumolysin 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 S1’s / myosin, mass c. 120 kD • ‘cross-bridge’ between myosin and actin filaments, thought to be source of force generation

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

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

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: S 15 nm S1 10 nm S2 5 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