Selected Examples of TEM Data Produced Using the Negative Staining Protocols

Negative staining using the droplet procedure on continuous carbon support films is widely applicable, indeed to almost all isolated biochemical and biological samples, and indeed solutions of synthetic polymers. Numerous examples of data could be given; for further possibilities the reader is directed to the extensive scientific literature and the author's book (2). The Escherichia coli chaperone GroEL complexed with the lower mass GroES to form the symmetrical/ellipsoidal complex is shown in Fig. 6. This specimen is negatively stained with 2% uranyl acetate on a continuous carbon support. Some chains of GroES-linked GroEL molecules are present, together with free GroES molecules. When trehalose is included in the negative stain solution, a thicker layer of dried stain and carbohydrate supports the biological material. This layer reduces flattening and enables the molecules to be more freely supported in varying angular orientations. This is shown in Fig. 7 for the elongated multidecamers and didecamers of keyhole limpet hemocyanin type 2, negatively stained with a mixture of 5% ammonium molybdate and 1% trehalose (pH 7.0). There is some evidence, by comparison with uranyl acetate, that ammonium molybdate might release carbon-adsorbed molecules, thereby increasing their freedom immediately before stain dries. When the single-droplet negative staining procedure is used with holey carbon support films, greater care has to be taken at the final stage to remove enough stain solution in order to produce a thin dried film. When this is achieved the holes of the specimen grid will be evenly spread with the biological sample embedded in the thin layer of dried stain + trehalose. Figure 8 shows an example of the decameric hemocyanin from Octopus dolfleini, spread across a holey carbon support film in the presence of 5% ammonium molybdate and

Groel Negative Stain
Fig. 6. The symmetrical complex of GroEL and GroES, negatively stained with 2% uranyl acetate after adsorption to a continuous carbon support film. Note the presence of some longer GroEL/GroES chains and free GroES molecules.

0.1% trehalose (pH 7.0). Inclusion of polyethylene glycol often promotes 2D crystal formation across the holes, achieved by the author for many different molecules and viruses. For tomato bushy stunt virus, the formation of 2D arrays and crystals is shown in Fig. 9, negatively stained with 5% ammonium molyb-date containing 1% trehalose and 0.1% PEG Mr 1000 (7).

The negative staining-carbon film procedure is particularly successful when used for the production of 2D protein and virus crystals. The example given in Fig. 10 is of the 20S proteasome from Thermoplasma acidophilum, in this case negatively stained finally with 2% ammonium molybdate rather than the more usually used uranyl acetate. Note the large 2D crystal with a few proteasome molecules missing from the lattice. For the E. coli chaperone GroEL, when 1 mM Mg-ATP is present the molecule adopts a unique 2D hexagonal lattice with a large central hexagonal space, that in the example shown (see Fig. 11) is filled with negative stain (uranyl acetate). The inset in Fig. 11, shows the 2D image reconstruction from this 2D crystal, revealing the manner in which the individual GroEL molecules are positioned within the hexagonal lattice.

Groel Micrograph

Fig. 7. Keyhole limpet hemocyanin (elongated KLH2 multidecamers and didecamers) negatively stained with 5% ammonium molybdate containing 1% trehalose on continuous carbon support films. This stain combination creates a relatively thick embedding layer that can support the molecules in varying orientations, as shown by the presence of the indistinct images of tilted molecules (left-hand panel). Bars,100 nm.

Fig. 7. Keyhole limpet hemocyanin (elongated KLH2 multidecamers and didecamers) negatively stained with 5% ammonium molybdate containing 1% trehalose on continuous carbon support films. This stain combination creates a relatively thick embedding layer that can support the molecules in varying orientations, as shown by the presence of the indistinct images of tilted molecules (left-hand panel). Bars,100 nm.

For fragile molecules that require the presence of a high concentration of glycerol for stability or are dramatically aggregated by uranyl acetate during the conventional droplet negative staining, the negative staining-carbon film procedure offers an alternative possibility. The enzyme tripeptidyl peptidase-II is in this category. In solution, tripeptidyl peptidase-II forms oligomers that generate elongated arc and double-bow complexes. The oligomeric intermediates that lead to the formation of these complexes are shown in Fig. 12.

The negative staining-carbon film procedure also can be used to produce cellular cleavage. In Fig. 13, an example is shown of a human erythrocyte wet-cleaved to reveal a single membrane layer, with the internal/cytoplasmic surface negatively stained with 2% uranyl acetate.

Cryonegative staining is likely to become an increasingly used technique for intermedate resolution molecular and virus studies (i.e., slightly above 1 nm), as it provides the benefits of freeze-preservation combined with the benefits of increased image contrast imparted by the negative stain. Figure 14 shows an electron micrograph of cryo-negatively stained keyhole limpet hemocyanin

Molybdate Tem
Fig. 8. Octopus hemocyanin negatively stained with 5% ammonium molybdate + 1% trehalose, spread unsupported across a hole in a holey carbon support film. Note the presence of the single ring-like end-on hemocyanin decamers and small stacks of side-on decamers (arrowheads).
Ammonium Molybdate And Negative Stain

Fig. 9. Two examples of tomato bushy stunt virus negatively stained with 5% ammonium molybdate containing 1% trehalose and 0.1% polyethylene glycol (Mr 1000) across holes in a holey carbon support film. The presence of the polyethylene glycol has induced the formation of a disorganized 2D array (A) and a 2D crystal with a hexagonal lattice (B). Modified from Harris and Scheffler (7).

Fig. 9. Two examples of tomato bushy stunt virus negatively stained with 5% ammonium molybdate containing 1% trehalose and 0.1% polyethylene glycol (Mr 1000) across holes in a holey carbon support film. The presence of the polyethylene glycol has induced the formation of a disorganized 2D array (A) and a 2D crystal with a hexagonal lattice (B). Modified from Harris and Scheffler (7).

Crystals Membrane Proteins

Fig. 10. A 2D crystal of the 20S proteasome from Thermoplasma acidophilum produced by the negative staining-carbon film procedure, with 2% ammonium molybdate (pH 7) as the final negative stain. Image processing suggests that the unit cell may contain six molecules because of the specific rotational orientation of the heptameric proteasome within the crystal (modifed from Harris [2]). Note the absence of individual and small groups of molecules from the 2D crystal lattice (arrowheads).

Fig. 10. A 2D crystal of the 20S proteasome from Thermoplasma acidophilum produced by the negative staining-carbon film procedure, with 2% ammonium molybdate (pH 7) as the final negative stain. Image processing suggests that the unit cell may contain six molecules because of the specific rotational orientation of the heptameric proteasome within the crystal (modifed from Harris [2]). Note the absence of individual and small groups of molecules from the 2D crystal lattice (arrowheads).

type 1 (KLH1) reconstituted from the subunit, with many didcecamers present at different angular orientations. Also present are single KLH1 decamers and tubular polymers. The somewhat-superior molecular detail here should be compared with that shown in Figs. 7 and 8.

Negative staining of immunologically and affinity-labeled biological samples is likely to be of increasing significance in the years ahead, as more epitope-

Structure Nuclear Pore

Fig. 11. A complex honeycomb 2D hexagonal lattice the E. coli protein GroEL in the presence of 1 mM Mg-ATP, prepared by the negative staining-carbon film procedure. The inset shows a reconstruction from the 2D crystal, which reveals the complex linkage of the individual cylindrical GroEL molecules forming the hexagonal lattice. This hexagonal 2D lattice was never encountered in the absence of ATP, which is in accord with evidence from others showing that a significant shape change is induced when ATP binds to GroEL.

Fig. 11. A complex honeycomb 2D hexagonal lattice the E. coli protein GroEL in the presence of 1 mM Mg-ATP, prepared by the negative staining-carbon film procedure. The inset shows a reconstruction from the 2D crystal, which reveals the complex linkage of the individual cylindrical GroEL molecules forming the hexagonal lattice. This hexagonal 2D lattice was never encountered in the absence of ATP, which is in accord with evidence from others showing that a significant shape change is induced when ATP binds to GroEL.

specific and residue-specific labels become available. Two examples of "in solution" immunonegative staining will be given. An immune complex of KLH2 with a monoclonal IgG directed against an epitope on the end of the molecule is shown in Fig. 15, negatively stained on a continuous carbon support film by 5% ammonium molybdate containing 1% trehalose. The molecules are linked to form chains, with one or more IgG molecules forming the bridges between the ends of the molluscan hemocyanin didecamer. When a KLH2 decamer ends a chain, further extension is not possible as the required epitope is not located at this position. If a biotinylated component is present within a biological molecule, subsequent labeling with streptavidin-gold is possible. Figure 16 shows an example of cholesterol microcrystals with a biotinylated mutant streptolysin-O molecule bound onto their surface. In solution, streptavidin conjugated 5-nm gold particles bind to the biotin, and negative staining with 2% ammonium molybdate shows convincing labeling at the edges and surface of the cholesterol microcrystals. A similar approach can be performed at a higher-resolution level if a His-tagged component is present by using a nickel-complexed gold cluster. When immunolabeling is performed on biological particles already adsorbed onto a

Igg Negative Stain

Fig. 12. Human erythrocyte tripeptidyl peptidase-II negatively stained with 2% ura-nyl acetate, by the negative staining-carbon film procedure (10). Conventional droplet negative staining of this enzyme complex with uranyl acetate on a carbon support film induced unacceptable aggregation. This specimen was produced directly from a protective 30% glycerol solution, by in vacuo drying followed by carbon coating and floating off onto negative stain. The high molecular mass tripeptidyl peptidase complex is revealed as elongated arc-like oligomers of the protein (arrowheads), alongside smaller oligomers.

Fig. 12. Human erythrocyte tripeptidyl peptidase-II negatively stained with 2% ura-nyl acetate, by the negative staining-carbon film procedure (10). Conventional droplet negative staining of this enzyme complex with uranyl acetate on a carbon support film induced unacceptable aggregation. This specimen was produced directly from a protective 30% glycerol solution, by in vacuo drying followed by carbon coating and floating off onto negative stain. The high molecular mass tripeptidyl peptidase complex is revealed as elongated arc-like oligomers of the protein (arrowheads), alongside smaller oligomers.

carbon-plastic support film, considerable possibilities exist for antigen localization. Micronemes isolated from the parasite Cryptosporidium parvum are very fragile, and even after stablization with 0.05% v/v glutaraldehyde some show damage. In Fig. 17, micronemes are shown, following on-grid labeling with a mouse monoclonal antibody and a secondary IgG complexed to 5 nm gold, negatively stained with 2% ammonium molybdate. This IgG is directed to a protein epitope that is present within the micronemes. Thus, only the content of damaged micronemes is labelled. The smooth-surfaced intact stain-excluding micronemes show no labeling.

Negative staining of dynamic biological systems has considerable potential, as long as the time period under consideration is in excess of approx 2 min. The formation of the dodecahedral macromolecular complex containing 12 toroidal 218-kDa human erythrocyte peroxiredoxin-2 decamers has been attempted by the author using negative staining on holey carbon support films in the presence of PEG. If the enzyme sample is incubated with PEG for a few minutes and then EM specimens produced with PEG present at every washing and negative staining

Electron Microscopy Protocol Pictures
Fig. 13. A single layer of human erythrocyte membrane produced by cell cleavage during the negative staining-carbon film procedure (10). The membrane has been negatively stained with uranyl acetate, revealing the uneven spectrin network on the cyto-plasmic surface.

stage, dodecahedral peroxiredoxin-2 complexes can be shown by TEM to have formed (see Fig. 18). The complete standardization of this sterically interesting macromolecular assembly, which is induced instead of 2D crystal formation in this instance, is currently under investigation.

The interaction time of antigen-antibody and biotin-streptavidin can often be assessed using negative staining. For the biotin-streptavidin reaction, this has been found to be rapid, by showing the formation of streptavidin-labeled tubules of synthetic DNA formed from oligonucleotides, one of which was biotinylated (16), after an incubation period of only 30 min (see Fig. 19). This specimen was spread across a holey carbon support film and was negatively stained with 5% w/v ammonium molybdate containing 0.1% w/v trehalose. Similarly, the interaction time of the Vibrio cholerae cytolysin with cholesterol micro-crystals occurs at the crystal bilayer edges within a few minutes, but extends onto the planar surface over a period of hours (17). The images of this material, shown in Fig. 20, were negatively stained with 2% ammonium molybdate on a carbon support film.

Groels Negative Stain
Fig. 14. Reassociated keyhole limpet hemocyanin type 1 revealed by cryo-negative staining with 15% w/v ammonium molybdate. Note the presence of decamers (arrowheads), didecamers and longer tubular structures (courtesy of Marc Adrian).
Molybdate Tem

Fig. 15. An immune complex containing IgG-linked keyhole limpet hemocyanin type 2 molecules. Note the single decamers (large arrowheads) and multiple IgG molecules linking the hemocyanin molecules (small arrowheads). Negatively stained on a continuous carbon film with 5% ammonium molybdate containing 1% trehalose.

Fig. 15. An immune complex containing IgG-linked keyhole limpet hemocyanin type 2 molecules. Note the single decamers (large arrowheads) and multiple IgG molecules linking the hemocyanin molecules (small arrowheads). Negatively stained on a continuous carbon film with 5% ammonium molybdate containing 1% trehalose.

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