Negative Staining-carbon Film Research Articles

Trichomonas vaginalis: observation of coexistence of multiple viruses in the same isolate.

Trichomonas vaginalis is a flagellated, parasitic protozoan that inhabits the urogenital tract of humans. Some isolates of T. vaginalis are infected with a double-stranded RNA (dsRNA) virus, which was described in the literature as homogeneous icosahedral viral particles with an isometric symmetry and 33 nm in diameter. This study examined in detail the viral particles in T. vaginalis isolate 347 and describes a heterogeneous population of viral particles. The different dsRNA viruses were only observed after a change in the technique. The sample was prepared by the negative staining carbon-film method directly onto freshly cleft mica. The detected viruses ranged in size from 33 to 200 nm. Among the shapes observed were filamentous, cylindrical, and spherical particles. These results show that T. vaginalis may be a reservoir for several different dsRNA viruses simultaneously.

Trichomonas vaginalis: observation of coexistence of multiple viruses in the same isolate

Trichomonas vaginalis is a flagellated, parasitic protozoan that inhabits the urogenital tract of humans. Some isolates of T. vaginalis are infected with a double-stranded RNA (dsRNA) virus, which was described in the literature as homogeneous icosahedral viral particles with an isometric symmetry and 33 nm in diameter. This study examined in detail the viral particles in T. vaginalis isolate 347 and describes a heterogeneous population of viral particles. The different dsRNA viruses were only observed after a change in the technique. The sample was prepared by the negative staining carbon-film method directly onto freshly cleft mica. The detected viruses ranged in size from 33 to 200 nm. Among the shapes observed were filamentous, cylindrical, and spherical particles. These results show that T. vaginalis may be a reservoir for several different dsRNA viruses simultaneously.

Virus in Trichomonas—an ultrastructural study

Trichomonas vaginalis is a flagellated, parasitic protozoan that inhabits the urogenital tract of humans. Approximately one-half of isolates of T. vaginalis are infected with a double-stranded (ds) RNA virus, which was described in the literature as a homogeneous population of icosahedral virus with isometric symmetry and 33 nm in diameter. The present study describes the heterogeneous virus population found in T. vaginalis isolate 347. This population comprises different virus sizes (33–200 nm) and shape (filamentous, cylindrical, and spherical particles). These observations were made in CsCl-purified virus fractions as well as the thin sections of parasites. Some viruses were only observed after slight changes in the technique where the sample was prepared by the negative staining carbon-film method directly onto freshly cleft mica. The VLPs were found in the cytoplasm closely associated with the Golgi complex, with some VLPs budding from the Golgi, and other VLPs were detected adjacent to the plasma membrane. Unidentified cytoplasmic inclusions were observed in the region close to the VLPs and Golgi. These results indicate that T. vaginalis organisms may be infected with different dsRNA viruses simultaneously and suggest that T. vaginalis may be a reservoir for several viruses. We also showed some steps in the route of T. vaginalis virus and some aspects of the cytopathology of this infection. Purified VLPs were transfected to virus-free T. vaginalis isolates. Our results demonstrate that TVV attach and penetrate into trichomonads through endocytic coated pits and are maintained within vacuoles during batch culture for several daily passages. Immediately after virus transfection, many cells were lysed, whereas some intact reminiscent cells were recruited forming large clusters. Virus particles were found outside the cells, and in coated pits, within vacuoles in the cytoplasm, and infrequently within the nucleus. The Golgi complex showed changes in its electron density and in the cisternae structure. In lysed cells, virus particles were clearly seen over the residual membranes.

Transmission electron microscopical studies on some haemolymph proteins from the marine polychaete Nereis virens

The hexagonal bilayer haemoglobin molecule from Nereis virens has been investigated in a comparative study using several different negative stain electron microscopical specimen preparations (i.e. by conventional adsorption to continuous carbon support films, by the negative staining-carbon film technique and by negative staining across the holes of holey carbon support films with air-drying and rapid freezing/cryo-negative staining). The benefits and limitations of these different approaches are indicated, with the overall conclusion that negative staining with ammonium molybdate across holes creates the best possibilities for molecular imaging, and also has the potential for the creation of two-dimensional (2D) crystals/arrays at the fluid–air interface. Of the different negative staining procedures presented, cryo-negative staining reveals the greatest details of N. virens haemoglobin. This is exemplified by the direct visualisation of the central linker-assembly within the haemoglobin molecule, a structural feature less clearly defined by the other negative staining techniques. A discoidal lipoprotein molecule (diameter 30–60 nm; thickness ca 8 nm) has been detected in N. virens, which represents the first documented account of an annelid haemolymph lipoprotein. The biological implications of this lipoprotein for lipid transport remain to be established. The presence of a low concentration of ferritin molecules in N. virens haemolymph is also shown, assisted by the formation of small 2D ferritin arrays in negatively stained specimens prepared across holes.

Keyhole limpet hemocyanin (KLH), I: Reassociation from Immucothel® followed by separation of KLH1 and KLH2

Studies of keyhole limpet hemocyanin (KLH) normally require purification of functional complexes directly from living animals. An alternative procedure is described wherein a commercial preparation of KLH which is fully dissociated into its subunits (Immucothel®, biosyn Arzneimittel GmbH) is reassociated in the presence of a high concentration of calcium and magnesium. The reassociation products, when observed by electron microscopy, consist of didecamers, multidecamers and flexible tubules of varying length. The two forms of KLH described previously and designated KLH1 and KLH2, are present in the reassocated mixture as homo-oligomers/polymers and can be separated by selective dissociation of the KLH2 by treatment with 1% ammonium molybdate-0.2% PEG at pH 5.7, followed by gel filtration chromatography in this solution. In addition to discrete elution peaks containing didecameric KLH1 and dissociated subunits of KLH2, a leading peak contains a tubular/polymeric form of KLH1, not previously described. Under negative staining conditions designed specifically for the creation of 2-dimensional crystals on mica (the negative staining-carbon film procedure), this tubular form of KLH1 can be transformed into a larger diameter multidecameric form, again not previously described for KLH1. The purified KLH2 peak is indistinguishable from subunit material prepared from living animals. Thus, Immucothel® appears to provide a standardized source of subunits suitable for biochemical and structural studies on the two types of KLH.

Electron Microscopy of the GroEL-GroES Filament

Electron microscopy of a filamentous complex of GroEL and GroES has been performed on negatively stained specimens. The filaments have been formed when GroEL and GroES at relatively low molar ratios (e.g., 1:1 to 1:3) are incubated at room temperature in the presence of magnesium and ATP. At higher molar ratio, the symmetrical GroEL-GroES complex predominates. Within the GroEL-GroES filaments, the same structural rearrangements of GroEL were observed as in the "bullet-shaped" or symmetrical GroEL-GroES complexes. In the absence of GroES, GroEL molecules have no tendency to form linear filaments. The sharing of one GroES by two GroEL molecules might indicate a high plasticity of the GroEL-GroES complex. Preliminary Fourier analysis of filaments of increasing length indicates that they possess an inherent helicity. Using the negative staining-carbon film procedure, two-dimensional (2-D) crystal nucleation induced by ammonium molybdate-polyethylene glycol can create undulatory rows of side-on GroEL molecules, which are able extend as a p2 2-D crystal form, readily distinguishable from the filamentous GroEL-GroES aggregates. Crystallographic image processing indicates that the GroEL 2 × 7-mer itself possesses a dyad screw axis, in accord with existing higher resolution X-ray structure data.

Human Erythrocyte Catalase: 2-D Crystal Nucleation and Production of Multiple Crystal Forms

Negatively stained electron microscope images are presented, showing the nucleation of two-dimensional (2-D) crystals of human erythrocyte catalase produced on mica by the negative staining-carbon film technique. Examples of the formation of partially ordered 2-D arrays and more ordered 2-D crystals are shown and the conditions required for the production of large well-ordered 2-D crystals discussed. The structural transformation of one flexuous 2-D paracrystal into a p2 2-D crystal is considered. The crystallographic 2-D image average of this p2 crystal form is presented (lattice parameters a = 9.0 nm, b = 18.6 nm; γ= 90-8°). It is shown that transmission electron microscopy provides the possibility of defining 2-D crystal nucleation, growth of intermediate forms, and low-resolution crystallographic structural analysis of 2-D crystals of human erythrocyte catalase. Comparison of the various electron microscopical negatively stained images with the peptide backbone of the X-ray structure of bovine liver catalase at different tilt and rotation positions correlates with and emphasizes the multiple intermolecular contacts and orientations that can be adopted by human erythrocyte catalase, leading to various 2-D arrays and 2-D crystals. Alignment of the surface groups involved in the protein-protein interactions that occur during 2-D crystal nucleation and crystal growth may ultimately be determined. From this approach, when taken together with detailed consideration of protein-solvent and protein-solute electrostatic interactions in solution, and at the fluid-air interface, it is considered that a more general theory of crystal nucleation and growth may eventually emerge.

Keyhole limpet haemocyanin (KLH): Purification of intact KLH1 through selective dissociation of KLH2

Keyhole limpet haemocyanin (KLH) from almost all newly captive animals contains a mixture of KLH1 and KLH2. We show that the dissociation of KLH2 can be produced during EM specimen preparation by the negative staining-carbon film (NS-CF) procedure and in solution by ammonium molybdate-PEG solutions at slightly acidic pHs. The KLH2 multidecamers split apart in the pH range 7.5-6.5 and in the pH range 6.5-6.0 the individual decamers break open and start to dissociate. At pH 5.9 the dissociation of KLH2 yields predominantly a mixture of single subunits and what appear to be subunit dimers. Over the pH range 7.0-5.7 the KLH1 didecamer remains stable. Separation of intact KLH1, in the form of didecamers and small clusters of PEG at pH 5.9 (pH 6.2 at 4°C). Assessment of the achieved separation of KLH1 and KLH2 was monitored by gel electrophoresis under native conditions and by electron microscopy of negatively stained specimens. Integrity of the multi-domain KLH1 and KLH2 subunits was assessed by SDS-PAGE. The significance of this separation for the overall understanding of the two types of KLH is that it provides for the first time purified KLH1 didecamers. Reassociation of the pH 5.9 dissociated KLH2 was achieved by prolonged dialysis against calcium-supplemented stabilizing buffer at pH 7.2 and resulted in multidecamers and slightly smaller diameter helical tubes.

Transmission Electron Microscopy of GroEL, GroES, and the Symmetrical GroEL/ES Complex

Two new 2-D crystal forms of the Escherichia coli chaperone GroEL (cpn60) 2 × 7-mer have been produced using the negative staining-carbon film (NS-CF) technique. These 2-D crystals, which contain the cylindrical GroEL in side-on and end-on orientations, both possess p21 symmetry, with two molecules in the respective unit cells. The crystallographically averaged images correlate well with those obtained by other authors from single particle analysis of GroEL and our own previous crystallographic analysis. 2-D crystallization of the smaller chaperone GroES (cpn10) 7-mer has also been achieved using the NS-CF technique. Crystallographically averaged images of GroES single particle images indicate considerable variation in molecular shape, which is most likely due to varying molecular orientation on the carbon support film. The quaternary structure of GroES does, nevertheless, approximate to a ring-like shape. The complex formed by GroEL and GroES in the presence of ATP at room temperature has been shown to possess a symmetrical hollow ellipsoidal conformation. This symmetrical complex forms in the presence of a 2:1 or greater molar ratio of GroES:GroEL. At lower molar ratios linear chains of GroEL form, apparently linked by GroES in a 1:1 manner, which provide supportive evidence for the ability of both ends of the GroEL cylinder to interact with GroES. The apparent discrepancy between our data and that of other groups who have described an asymmetrical "bullet-shaped" (holo-chaperone) GroEL/ES complex is discussed in detail.

Negative staining: A brief assessment of current technical benefits, limitations and future possibilities

Some personal opinions are expressed regarding the future of negative staining in biomolecular electron microscopy. The situation of varying depth negative stain and the consequent partial staining of biomolecules, the possible benefits that might be gained from trehalose-negative stain combinations, the improved stain spreading and permeation by the inclusion of surfactants and further applications of the negative staining-carbon film procedure are all discussed in detail. Examples are provided from several different macromolecules, including keyhole limpet haemocyanin, Nereis haemoglobin and the E. coli GroEL/ES complex. The positive message is given that with continuing technical development, negative staining still has much to offer.

Electron Microscopy of Human Erythrocyte Catalase: New Two-Dimensional Crystal Forms

Using the mica-spreading "negative staining-carbon film" procedure, human erythrocyte catalase has been shown to create a number of different periodic or crystalline two-dimensional (2-D) arrays which differ in the arrangement of molecules in the repeating units and the lattice type. Digital image processing has been performed with a 2-D array which contains regularly arranged "undulating" rows of molecules and also with a 2-D crystal form, exhibiting pgg (p22121) symmetry and lattice parameters of a = 12.7 nm, b = 44 nm, and γ= 92°. The data are compared with our previous analysis of a different human erythrocyte catalase 2-D crystal, and the effect of partial-depth negative staining occurring in all the various 2-D forms is discussed.

Ubiquitous soluble Mg 2+-ATPase complex : A structural study

We have performed a detailed structural analysis of the soluble Mg 2+-ATPase complex purified from Xenopus laevis ovary, which is an abundant and ubiquitous homo-oligomeric protein complex located in the nucleus and in the cytoplasm, belonging to a novel multigene-family of putative Mg 2+-ATPases. Enzyme activity staining after non-denaturing polyacrylamide gel electrophoresis revealed that Mg 2+-ATPase activity of the native protein is dependent on oligomerization and could not be detected in dissociated subunits. For the native protein a sedimentation coefficient of 15·3 S and a corresponding relative molecular mass of 612,000 was determined by analytical ultracentrifugation and a relative molecular mass of 590,000 was estimated from scanning transmission electron microscopy, supporting our previous conclusion that the oligomer comprises six 97,000 M r subunits. Conventional electron microscopy of negatively stained specimens revealed the Mg 2+-ATPase complex to be a hexagonal molecule in its favoured “end-on” projection and a double-banded molecule in its “side-on” projection (approx. 12 nm diameter; approx. 9 nm height). In addition, dimerized complexes could be observed in negatively stained specimens, yielding pronounced hexameric images and four-banded images in their end-on and side-on orientations, respectively (approx. 12 nm diameter; approx. 18·5 nm height). Two-dimensional (2D = mono-molecular) crystals have been produced from the dimerized complexes by the negative staining carbon film technique. Hexagonal crystals with a p6 plane group symmetry were obtained from molecules in their end-on orientation and longitudinal arrays with a p2 symmetry from complexes in their side-on orientation. A low-resolution molecular model of the native protein, derived from averages of these two 2D crystals, is presented. From our results we propose oligomerization as an inherent structural principle of organization for this whole newly defined Mg 2+-ATPase multigene-family, that includes such seemingly diverse functionally defined proteins as mammalian and yeast “vesicle fusion” and “peroxisome assembly” proteins and the product of the yeast cell cycle gene CDC48.

Two-dimensional crystallization, transmission electron microscopy and image processing of keyhole Limpet Haemocyanin (KLH)

KLH, the respiratory protein haemocyanin of the keyhole limpet Megathura crenulata, has been obtained as a high g pellet from the cell-free haemolymph and purified by gel permeation chromatography on a Biogel A15m column. The leading major protein peak eluted has been found to contain haemocyanin multi-decamers, followed by a second major peak containing single di-decamers, with small amounts of decamer and partly dissociated material following in the later fractions. The purified KLH di-decamer has been for two- dimensional crystallization studies with the negative staining-carbon film technique, in the presence of polyethylene glycol. In the side-on orientation, KLH has been found to produce two-dimensional crystals with a half-molecule linear displacement in consecutive rows. This is shown to be due to a specific association and two-dimensional crystal nucleation of the molecules in this arrangement. When oriented end-on, KLH has been found to form close- packed hexagonal monomolecular arrays which are not truly crystalline. This is because the five-fold rotational symmetry of the cylindrical macromolecule is not readily compatible with the hexagonal molecular packing. Computer-processed averaged images have been produced from the side-on KLH two-dimensional crystals and the end-on arrays, which reveal the principal molecular features of this homo-oligomeric protein complex to a resolution of ca 2.7 nm.

Negative staining-carbon film technique: New cellular and molecular applications

Two techniques are presented which extend the original negative staining-carbon film technique into new areas of cellular and molecular application. These relate (1) to the production of negatively stained specimens of single-layer plasma membrane split from intact cells during the overall procedure that are negatively stained from the cytoplasmic face and (2) to the production of negatively stained specimens directly from glycerol-containing protein solutions, membrane or viral suspensions. In both cases in vacuo drying onto mica from glycerol is performed, prior to deposition of a carbon film. (For the cellular technique, freshly cleaved mica is firstly rendered positively charged by immersion in Alcian blue.) This is followed by release of the carbon film plus adsorbed membrane or protein by floating onto water, with subsequent negative staining. Selected preliminary applications using human erythrocyte membrane and the high molecular weight (native) human erythrocyte tripeptidyl peptidase-II complex are given and considered speculation as to the future application of the techniques is provided.

The negative staining-carbon film procedure: Technical considerations and a survey of macromolecular applications

The development and initial virological applications of the negative staining-carbon film (NS-CF) procedure are described and followed by a survey of some recent macromolecular applications of the technique. Electron microscopical data are shown which indicate the capability of the technique for the production of two-dimensional (2-D) crystals from several different soluble proteins (human erythrocyte catalase, human erythrocyte, Xenopus and Thermoplasma acidophilum cylindrin/multicatalytic proteinase, yeast glutamine synthetase and keyhole limpet haemocyanin). Instances in which ordered two-dimensional close-packing of molecules is produced, rather than true two-dimensional crystallization, are also indicated. In addition, data obtained from the application of the NS-CF procedure to a number of fibrillar proteins are briefly presented. Examples include bovine lens vimentin, thrombin-cleaved vimentin and bacterially expressed Xenopus lamin-A. These elongated proteins form higher order filamentous structures, networks and, in the case of lamin-A, large paracrystalline structures. A general discussion of the achievements and future potential of the NS-CF procedure is presented.

Electron microscopical and biochemical studies on the oligomeric states of human erythrocyte tripeptidyl peptidase—II

The oligomeric structure of native and low ionic strength-dissociated human erythrocyte tripeptidyl peptidase II (TPP-II) have been investigated by PAGE (without SDS) and transmission electron microscopy. The native TPP-II complex has a very high molecular mass (>106) and has a ‘double bow’ or short double helical conformation, 50 nm in length and 20 nm wide. This complex dissociates into a range of smaller oligomers upon dialysis against a dilute Tris-HCl buffer, with an Mr 270,000 dimer having some tendency to predominate during short (1 to 4 h) dialysis times. Small 2-dimensional arrays of the dissociation products have been produced using the negative staining-carbon film technique when polythylene glycol (Mr 10,000) is included, within which the Mr 270,000 dimer of TPP-II appears to be the repeating unit.

Transmission electron microscopic studies on the quaternary structure of human erythrocyte catalase

Two-dimensional crystals of the M r 256,000 human erythrocyte catalase with typically a × b = 8.44 × 8.43 ± 0.5 nm (α=81.6°) have been prepared by the negative staining-carbon film technique, in the presence of 0.05% (w/v) polyethylene glycol ( M r 10,000). In the absence of polyethylene glycol no crystal were detected. One predominant molecular projection arbritarily defined as the face-on-view, is present within the two-dimensional crystals, although the enzyme also has a tendency to form rows of molecules. Assuming a projection normal to the latter (side-on-view), the human erythrocyte catalase is approx 6 nm in height. Digital Fourier peak filtering has been performed on the two-dimensional crystals. Averaged unit cell projections revealed the four subunits of the molecule. Projections of single molecules provided further clues about the quarternary structure of human erythrocyte catalase. In conclusion, the three-dimensional structure of human erythrocyte catalase at low resolution ( ca 2 nm) resembles a flattened tetrahedron with its two principal axes being not orthogonal to each other and with its four edges corresponding to the distal ends of two pairs of subunits comprising two separate planes. Each subunit has a pseudo-spherical appearance with ellipsoidal domains towards the periphery. In a face-on projection the long axes of these domains are not parallel with the two principal axes of the tetrahedron, thus giving rise to a chirality. Owing to the proposed three-dimensional structure, two of the subunits predominate in the averaged face-on projection of the molecule.

α-Mannosidase from jack bean ( Canavalia Ensiformis): Negative staining of single molecules and paracrystalline forms

Purified jack bean α-mannosidase has been shown by conventional negative staining, using sodium phosphotungstate, to be a dimeric molecule, length ca 11.5 nm and width ca 3 nm. Electrophoretic data suggests that the dimeric molecule has a M r of ca 400,000 and that it contains two subunits of M r ca 135,000. It is proposed that the stable dimeric state of the molecule has the conformation (αβ) 2. Trace quantities of a tetrameric form are, however, detected by both electron microscopy and electrophoresis. Attempts to crystallize α-mannosidase in the presence of ethanol have led to the production of irregular molecular aggregates of varying size, but prolonged storage in the presence of 80% (v/v) ethanol produced some aggregates which exhibit quasi-crystallinity. The instability of the crystalline particles during the negative staining procedure presents a problem. Application of the negative staining-carbon film technique has been found to produce ca 10 nm rodlike structures, which tend to be oriented randomly, relative to one another. However, if polyethylene glycol-1000 (0.05%) is included before the mixture of enzyme and ammonium molybdate is allowed to dry onto the mica surface, the formation of two-dimensional paracrystals composed of a number of parallel 10 nm rods is induced. Under these conditions the 10 nm rods are shown to have a greater amount of negative stain located in a central channel than is the case in the absence of polyethylene glycol-1000.

Electron microscopy of human hepatitis B virus cores by negative staining-carbon film technique.

The ultrastructure of the nucleocapsid component of human hepatitis B virus (core particle) was studied by negative staining-carbon film technique. Using this method an improved image of core particles was obtained in respect of resolution and contrast. Two-dimensional crystalline arrays of core particles were formed in vitro. Under these arrays the distance between the particle centers was 28.3 nm, corresponding to the capsid diameter, when analyzed through optical diffraction patterns. Positively stained images of these arrays revealed that core particles contain an electron-dense center of nucleoid-like area about 21 nm in diameter. The capsid surface rarely exhibited small capsomeres, ie, small spheres or ring-like structures measuring 4.0-4.2 nm. From the dimension of these structures and the analysis by Markham's rotational technique, it was suggested that each of these capsomeres is an individual subunit (monomer) and 180 of these subunits build up the core particle capsid according to the icosahedral symmetry (T = 3), but not clustering into distinct morphological features.

The production of paracrystalline two-dimensional monolayers of purified protein molecules

The application of the negative staining-carbon film technique to a range of different high molecular weight protein molecules is described. The influence of symmetrical and asymmetrical quaternary conformation of the protein on the success as previously described fo icosahedral virus particles by Horne and his colleagues, in much the same manner as previously described for icosahedral virus particles by Horne and his colleagues, in readily producing ordered two-dimensional monolayer arrays. Nevertheless, random and ordered multi-layer regions of apoferritin are also encountered. The other proteins studied, namely, human erythrocyte cylindrin and torin, the Bordetella pertussis ‘22S antigen’ and Escherichia coli glutamine synthetase, all of which have asymmetrical quaternary conformations based on the hollow cylinder model, produce ordered arrays to a lesser extent. Factors such as the preferential orientation of the protein on the mica and carbon film surface, the inter-molecular interactions which may tend to assist or prevent ordered monolayer formation, and the protein concentration will all influence the success achieved. The problems encountered can be approached by slight variation of the technique with respect to the negative stains used, their pH and concentration, the temperature and the length of time during which the protein solution dries on to the mica surface.