R.W. Horne

A negative staining—carbon film technique for studying viruses in the electron microscope: I. Preparative procedures for examining icasahedral and filamentous viruses

Summary A simple technique has been developed for the preparation of highly concentrated or crystalline suspensions of viruses in the presence of negative stains. Several icosahedral and filamentous viruses have been studied in concentrations from 1.0–20 mg/ml. The technique has several advantages over conventional negative staining procedures by forming two-dimensional and three-dimensional crystalline arrays of highly concentrated viruses. The packing arrangements of the virus in the crystalline arrays can be varied according to the type of negative stain and pH used during the preparation. High resolution electron micrographs of material prepared by the method have been analysed by optical diffraction procedures. The relative thickness of the carbon supporting film in relation to the physical dimensions of viruses is considerably improved.

The ultrastructural organization of elastin

The ultrastructural organization of elastin has been studied by electron microscopy of negatively stained specimens. Fine suspensions of elastin from ligamentum nuchae were obtained by mechanical stirring or by sonication and subsequently were purified under carefully controlled procedures. In the presence of various negative stains, the elastin fibers were seen to be composed of a network of branched bundles possessing a wide range of diameters. High resolution micrographs revealed that the fibers consisted of slender filaments of about 30–40 A diameter arranged roughly parallel to the fiber long axis. A regular periodicity of about 40 A along the filaments was also resolved. The images from negatively stained specimens are discussed in relation to the structures seen in thin-sectioned material together with current views concerning the interpretation of the submicroscopic and molecular architecture of elastin.

The surface structure of spores and aerial mycelium in Streptomyces coelicolor

The fine structure of the cell envelopes of vegetative aerial hyphae and spores of Streptomyces coelicolor was studied by means of freeze-etching and negative staining. The walls of both hyphal cells and spores appeared smooth or slightly granular without any periodic substructure. The thin surface layer surrounding the cell wall consisted of a delicate sheet covered with pairs of longitudinally furrowed rodlets arranged in a mosaic pattern. The length of most of the rodlets as seen in freeze-etchings ranged from 60 to 150 nm, which is likely to be only part of their full length. The negative staining procedure disrupted the original rodlet pattern and revealed isolated rodlet units up to 450 nm long. It is discussed whether the pattern represents a genuine fibrous complex or if the rodlets are only thickenings or folds in a membrane, thus simulating interlacing fibers. The rodlets were not markedly affected by organic solvents, and it is suggested that the surface layer is not (or not only) composed of lipids as previously supposed.

Electron microscopy and optical diffraction of elastin

Abstract The ultrastructural organization of purified elastin has been studied with the aid of electron microscopy and optical diffraction. Elastin fibril bundles were observed to have a considerable range of diameters. The basic morphological component resolved in negatively stained preparations of elastin bundles was seen as a slender filament of undefined length arranged roughly parallel to the long fibre axis. The diameter of the filaments was 3–4nm with an average centre-to-centre distance of 5nm. A regular periodicity of about 4–4.5nm was resolved along the individual filaments. Evidence of ‘cross-bridging’ spaced at about 3–3.5nm along the filaments was also resolved in many electron micrographs. Analysis of the electron micrographs with the aid of optical diffraction showed spectra with an equatorial reflection of 5nm and weak near meridional spectra at 4nm. The electron microscope images when considered together with independent X-ray diffraction data, suggested a model for the elastin filament as being a rope-like structure of about 3.5–4.Onm dia and composed of paired sub-filaments of about 1.5nm dia. The proposed model also offered a possible explanation for the small changes observed in the axial periodicity along the elastin filaments, on the basis of their being in a stretched or relaxed state.

The assembly of papaya mosaic virus protein.

Abstract Protein isolated from papaya mosaic virus, which is a flexuous virus similar in morphology to potato virus X, will self-assemble into long helical particles in the absence of RNA. The significance of the conditions required for assembly are briefly considered.

A negative staining-carbon film technique for studying viruses in the electron microscope: III. The formation of two-dimensional and three-dimensional crystalline arrays of cowpea chlorotic mottle virus

The negative staining-carbon film technique was applied to purified and highly concentrated suspensions of cowpea chlorotic mottle virus (CCMV) wild type and a salt-stable mutant. Both two-dimensional and three-dimensional crystals were rapidly formed at the surface of mica using CCMV suspensions containing about 2.0 to 14.0 mg/ml of virus. When mixed with 3% ammonium molybdate at pH 5.2 the crystals contained CCMV particles predominantly packed in a hexagonal array at the mica surface. At pH 7.0, the crystals were observed to be predominantly in square arrays. Under the preparative conditions used it was possible to form crystalline sheets with sides of several micrometers and in some instances covering entire grid squares. The crystals were found to be ideal for producing low-angle electron diffraction patterns and optical diffraction patterns from the micrographs with spectra extending out to about 2.0 nm. Experiments carried out on CCMV wild type and the salt stable mutants also showed that rapid morphological degradation occurs following storage at 4°C. The viral components from the degraded virus were observed to influence both the formation of the crystals and resolution obtainable from the specimens.

A negative staining-carbon film technique for studying viruses in the electron microscope: II. Application to adenovirus type 5

Two-dimensional and three-dimensional crystalline arrays of adenovirus type 5 were rapidly formed in vitro in the presence of negative stains when applied according to the negative staining-carbon procedure. The adenovirus capsids within the crystal lattice were packed predominantly in hexagonal arrays. Capsids were observed to be packed in positions viewed along axes of threefold symmetry and in other lattice arrays showed capsids in twofold symmetry positions. These crystalline arrays were extended over areas of several hundred microns across and provided ideal specimens for both optical diffraction and low-angle electron diffraction studies. When the adenovirus preparations were subjected to different negative staining and pH conditions, the capsids were observed to disrupt gradually at the specimen support surface. The released components were studied at high resolution and the structural components associated with the hexon and penton capsomeres were resolved. A detailed scale model of the adenovirus capsid was constructed from the information contained in the electron micrographs and the model used to illustrate the possible packing arrangements of the icosahedral capsid and penton fibres in the observed crystalline lattice.

Reconstitution of potato virus X in vitro: II. Characterization of the reconstituted product

Abstract Rapid reconstitution in vitro of infectious potato virus X (PVX) was achieved in mixtures of coat protein subunits and RNA prepared by freezing and thawing purified virus in 2 M LiCl. No reconstitution was obtained with protein subunits prepared with CaCl 2 , pyridine, or sodium dodecyl sulphate. The reconstitution reaction occurred at 20°C but not at 4°C, and at low (10 m M ) but not at moderate (100 m M ) salt concentrations; the optimal pH was between 6.0 and 6.2 The structure of reconstituted PVX was shown by optical diffraction analysis of electron micrograph images to be the same as that of native virus. Both reconstituted and native PVX had a basic helical pitch of 3.65 nm that repeated every eight turns along the particle axis, interparticle spacings of 12.6 nm, and no evidence of an axial hole. Reconstitution of PVX in vitro was accompanied by reappearance of the antigenic specificity of intact virus particles. Reconstituted virus particles were heterogeneous in size, and the ribonuclease resistant infectivity was generally less than 10% that of native virus. This may result from trace amounts of ribonuclease contamination in the protein subunit preparations hydrolyzing the RNA before reconstitution of full-length particles occurred.

The formation of two-dimensional arrays of isometric plant viruses in the presence of polyethylene glycol

Abstract The formation of two-dimensional arrays of isometric plant viruses using the negative staining carbon-film technique has been extended to include experiments on the addition of polyethylene glycol (PEG). The original negative staining carbon-film method depended on the availability of freshly prepared highly concentrated plant virus suspensions, as material stored at 4°C from 24hr to several weeks resulted in a considerable reduction in the areas of crystalline arrays. The addition of PEG, mol. wt. 6000, to stored plant virus suspensions containing cowpea chlorotic mottle virus, broad bean mottle virus, turnip rosette virus and southern bean mosaic virus has resulted in the formation of extensive continuous areas of crystalline arrays.

The Structure of the Exosporium of a Pigmented Clostridium

Summary: Exosporia surrounding spores of a pigmented Clostridium showed a hexagonal periodicity when negatively stained and were composed of up to 10 lamellae, each 2.6 nm thick, with a centre-to-centre spacing of 5.2 nm. Optical diffraction spectra revealed that isolated fragments of single lamellae contained hexagonal arrays of subunits with a predominant spacing of 5.2 nm and that negatively stained fragments composed of several lamellae showed interference patterns generated by slightly displaced layers of the same hexagonal lattice.