Basic Electron Microscopy - University of Nottingham

Basic Electron Microscopy - University of Nottingham

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 the negative staining method the metal-shadowing method _

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 9 8.54 8.49 8.48

8 Rh(nm) D20w (x10-7) 7 6 5

5.40 3.91 4 3 2 2.60

2.55 ES EL ELES 2.51 ELES+ ATPgamma-s

2.41 ELES+ 0.5mMATP 2.20 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 Pneumolys in 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 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 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 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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