Structure of adsorbed fibrinogen obtained by scanning force microscopy

It is shown that scanning force microscopy (SFM), operated in the attractive mode, can be used to obtain high resolution pictures of adsorbed fibrinogen molecules on solid surfaces, without the need for staining or special microscope grids. SFM also reveals the three-dimensional structure of the adsorbed molecules. Two forms of adsorbed fibrinogen are demonstrated on hydrophobic silicone dioxide surfaces; a trinodular about 60 nm long and a globular with about a 40 nm diameter. Polymeric networks formed after storage of the surface with adsorbed fibrinogen in PBS for 11 days are also shown. The SFM-results for the trinodular structure suggest the existence of loops or peptide chains extending outside the basic structure of the fibrinogen molecule.     

Two-dimensional crystals of a membrane protein: arrangement of subunits within the crystal sheet

Two-dimensional crystals have been prepared from the photosynthetic reaction center of Rhodopseudomonas viridis. Filtered images of these crystals show individual subunits approximately 4.5 nm in diameter arranged at a center-to-center distance of 6.4 nm. Our previous studies suggested that each subunit within such a sheet corresponds to a single photosynthetic reaction center. Air-dried and freeze-etched shadowed preparations of the crystals yield images which are quite different from negatively stained material. Rotary-shadowed surfaces of the crystals show rows of wedge-shaped particles separated by 3 nm furrows. Two such wedge-shaped particles occupy the 12.1 X 12.9 nm area in which four negatively stained subunits are normally visualized. Close analysis of these shadowed pictures suggests that both the shadowed and negatively stained images can be accounted for by a single model of subunit arrangement within the crystal. Within each 12.1 X 12.9 nm unit cell, two subunits are placed near one surface of the sheet, and two others are near the other surface. All four subunits are visible in negative stain. When the surface is shadowed, only the two subunits which project above the surface of the sheet accumulate appreciable amounts of the heavy metal shadow. Because of their close position, one subunit shades the other, forming the wedge-shaped appearance characteristic of the crystal. The only arrangement consistent with both shadowed and negatively stained images is one in which the two raised subunits occupy positions at either end of a diagonal across the unit cell. The analysis of shadowed images indicates that the plane group of the crystals is P22(1)2(1).     

AN ultrastructural study of cross-bridge arrangement in the frog thigh muscle thick filament.

We have developed thick filament isolation methods that preserve the relaxed cross-bridge order of frog thick filaments such that the filaments can be analyzed by the convergent techniques of electron microscopy, optical diffraction, and computer image analysis. Images of the filaments shadowed by using either unidirectional shadowing or rotary shadowing show a series of subunits arranged along a series of right-handed near-helical strands that occur every 43 nm axially along the filament arms. Optical filtrations of images of these shadowed filaments show 4-5 subunits per half-turn of the strands, consistent with a three-stranded arrangement of the cross-bridges, thus supporting our earlier results from negative staining and computer-image analysis. The optical diffraction patterns of the shadowed filaments show a departure from the pattern expected for helical symmetry consistent with the presence of cylindrical symmetry and a departure of the cross-bridges from helical symmetry. We also describe a modified negative staining procedure that gives improved delineation of the cross-bridge arrangement. From analysis of micrographs of these negatively stained filament tilted about their long axes, we have computed a preliminary three-dimensional reconstruction of the filament that clearly confirms the three-stranded arrangement of the myosin heads.     

STRUCTURE OF ISOLATED PLANT GOLGI APPARATUS REVEALED BY NEGATIVE STAINING

Sucrose-gradient-purified dictyosomes of plant Golgi apparatus appear, after glutaraldehyde stabilization, as stacks of highly fenestrate and tubate cisternae when negatively stained with phosphotungstic acid, shadowed with heavy metal, or OsO₄-stained in thin section. The tubular proliferations (diameter 200 to 400 A) extend for several microns from the central region and are united at intervals into an anastomosing network. Associated with the tubules are two kinds of vesicles which are distinguishable on the basis of texture, size, shape, and staining characteristics. One vesicle type is rough-surfaced, nearly spherical, and of uniform dimensions (diameter approximately 600 A). Metal shadowing shows that these vesicles remain spherical after drying. The other vesicle type is smooth-surfaced and varies in both size and shape. Intercisternal elements are revealed, by negative staining, on the surface of internal cisternae after fragmentation of the dictyosome. The progressive differentiation of cisternae from the forming face to the maturing face is observed in thin sections of these isolated preparations. The morphological characteristics observed in negatively stained dictyosomes indicate regions of functional specialization within the dictyosome cisternae and reveal a dictyosome structure more extensive than that envisioned from sections.     

Intracellular structures of Mycoplasma pneumoniae revealed after membrane removal.

Mycoplasma pneumoniae was grown on Formvar- and carbon-coated electron microscope grids and treated with the nonionic detergent Triton X-100 to gently remove the membrane and cytoplasm. The detergent mixture was composed of 0.5% Triton X-100 in SSR-2 broth base. After this treatment, the grids were rinsed in a mixture of 0.1 M KCl, 5 mM MgCl2, and 6 mM potassium phosphate buffer (pH 7.05) and negatively stained with uranyl acetate. The Triton X-100-resistant remains of M. pneumoniae after gentle removal of the membrane and cytoplasm consisted of fibrous structures oriented similarly to the undisrupted cells. The thin fibers displayed a negative staining quality and diameter analogous to that of rabbit muscle F-actin. The fibrous moieties ended in rodlike condensations which appeared striated in negatively stained and shadowed preparations. These striations were regular, and the majority of rod structures had lengths of 220 to 300 nm and widths of 50 to 80 nm. Specific antibody to rabbit muscle actin, produced in guinea pigs, was used in indirect immunofluorescence of the M. pneumoniae colonies. Fluorescence was detected, with concentrations at the colony center and at the tips of filamentous cells.     

Molecular dimensions of the SS and FF phenotypes of human placental alkaline phosphatase: Rotary shadowing and negative staining electron microscope measurements

The two most common homologous phenotypes (SS and FF) of human placental alkaline phosphatase were purified and observed in the electron microscope by rotary shadowing and negative staining techniques. In the rotary shadowing technique, the molecules of the two phenotypes appeared to be approximately elliptical with slit-like structures in the center of the molecules, suggestive of the groove between two subunits. The dimensions of the rotary-shadowed molecules were calculated as 10.1 nm × 5.7 nm for SS and 10.1 nm × 5.6 nm for FF phenotypes. The negative staining technique delivered more fine detail of the molecules than rotary shadowing. The predominant shape of the molecules in this method appeared to be rectangular, with a longitudinal stain-filled groove and with each of the half molecules (presumably 65,000 M_r subunit) very often appearing bi-lobed. This accounts for the molecules which appear to have four pronounced electron-transparent regions. The dimensions of the negatively stained rectangular-shaped molecules were measured as 7.5 nm × 5.5 nm for SS and 7.0 nm × 5.4 nm for FF phenotypes. No significant difference in electron microscopic appearance between the SS and FF phenotypes were observed.     

Identification and mass analysis of human fibrinogen molecules and their domains by scanning transmission electron microscopy

The domainal substructure and molecular conformation of human fibrinogen have been investigated by evaluating scanning transmission electron microscopic images of freeze-dried or negatively contrasted native fibrinogen (fractions I-4 and I-9), glutaraldehyde-treated fibrinogen, or plasmic core fragments D₁ and E₂. Although some unstained freeze-dried native or glutaraldehyde-treated fibrinogen molecules were relatively compact and even occasionally spheroidal, typical images were elongated symmetrical tridomainal structures 460 Å ± 20 Å in length; frequently they were bent into a variety of elongated though non-linear arrangements. Their identification as monomolecular forms of fibrinogen by scanning transmission electron microscopic mass measurements resolved uncertainties relating to the identity of such objects as single molecules. The central domains of fraction I-4 molecules had a greater mass than those of fraction I-9 (1.01 × 10⁵M_rversus 7.5 × 10 M_r, respectively). This difference accounted for the observed mass difference between fraction I-4 and fraction I-9 molecules (i.e. 3.27 × 10⁵M_rversus 2.97 × 10⁵M_r, respectively) and suggested that the COOH-terminal region of the Aα chain (major portions of which are always absent from fraction I-9 molecules) is situated within the mass integration radius for the central domain. When the COOH-terminal region of the Aα chain was present it appeared in negative stain as a thread-like structure originating between the middle and outer domains and extending toward the central domain, sometimes appearing to wind around the long axis.The outer domains of negatively stained molecules resembled negatively stained images of fragment D₁ and could frequently be resolved into at least two discrete subdomains, forming an oblong structure usually canted at an angle of ～120 ° to 150 ° relative to the long axis. Our findings are consistent with prevailing tridomainal structural models of fibrinogen and suggest that these molecules are flexible and may exist in unfolded configurations, or as relatively compact, partially or completely folded forms.     

Fibronectin molecule visualized in electron microscopy: a long, thin, flexible strand

We have determined the structure of plasma fibronectin by electron microscopy of shadowed specimens. the 440,000 molecular weight, dimeric molecule appears to be a long, thin, highly flexible strand. The contour length of the most extended molecules is 160 nm, but a distribution of lengths down to 120 nm was observed, indicating flexibility in extension as well as in bending. The average diameter of the strand is 2 nm and there are no large globular domains. the large fragments produced by limited digestion with plasmin are not globular domains but are segments of the strand, whose length corresponds to the molecular weight of the polypeptide chain. We conclude that each polypeptide chain of the dimeric molecule spans half the length of the strand, with their carboxyl termini joined at the center of the strand and their amino termini at the ends. This model is supported by images of fibronectin-fibrinogen complexes, in which the fibrinogen is always attached to an end of the fibronectin strand.     

Quantitative analysis of the mechanism of negative staining with native collagen fibrils and polar tropomyosin paracrystals

The mechanism of formation of the negatively stained image in electron microscopy was infestigated with native collagen fibrils as a model. The negatively stained image was simulated from the primary structure by using the values of volume or bulkiness of each amino acid residue as a parameter for stain-excluding capacity. The pattern simulated from the bulkiness values gave an excellent fit with the negatively stained image. Since some contribution of positive staining components to negative staining has been suggested, positive staining with uranyl acetate was tested with various washing solutions of different pH. While acidic conditions did not produce any stained image, a positively stained image was easily obtained at alkaline pH. On the other hand, negatively stained images with stains of different charge character remained essentially the same as those obtained with acidic uranyl stains. It was concluded that the contribution of positive components to the negatively stained image is negligible under the conventional conditions for negative staining with uranyl acetate. In order to demonstrate the utility of the analytical method employing the values of "bulkiness," we studied the unknown molecular packing in the polar lead paracrystal of rabbit skeletal tropomyosin. Utilizing the primary sequence data for alpha-tropomyosin we successfully showed the polar paracrystal to be an array of molecules which are parallel and in register. Further, our analysis made it possible to deduce the position of a given residue in the negatively stained pattern of the polar paracrystal.     

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 Mr 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.     

Crystalline layer in Drosophila melanogaster eggshell: arrangement of components as revealed by negative staining and reconstruction

Eggshell formation in Drosophila melanogaster is used as a model system in studies of cellular differentiation. A detailed knowledge of eggshell structure is necessary for this purpose and also to permit correlation of eggshell structure with function. Unique among the eggshell layers, the innermost chorionic layer (ICL) was investigated by means of transmission electron microscopy of thin sections and whole mounts, utilizing conventional fixation. LaNO₃ impregnation and negative staining with uranyl acetate. Whole mount face views of negatively stained ICLs were processed by means of optical and computer reconstruction. The ICL, which almost fully covers the oocyte, consists of 4 5 bilaminar sublayers with a total thickness of 400–500 Å. It appears to be formed by crystallites 1– μm in size, containing roughtly spherical molecules which are 30 Å in diameter approximately. Each unit cell probably includes 8 molecules and also distinct vacant spaces, differing in size, ICL may be involved in the exchange of the respiratory gases during embryogenesis.     

Macromolecular structure and aggregation states of Helicobacter pylori urease.

Urease purified from Helicobacter pylori by differential ultracentrifugation and fast pressure liquid chromatography was composed of subunits with apparent molecular weights (MrS) of 66,000 and 30,000. Electron microscopy of this purified material demonstrated that it formed disc-shaped macromolecular aggregates that were approximately 13 nm in diameter and 3 nm thick. Images of both negatively stained and shadowed preparations indicated that the discs tended to stack to form pairs and then these pairs further aggregated to form four-disc stacks. This stacking of subunits explains the heterogeneity observed previously in the molecular weight of urease preparations. In some negatively stained preparations there were also some smaller (approximately 8-nm-diameter) annular units present, which may represent individual urease units or possibly an aggregate of one of the two subunits from which urease is constructed.     

The molecular architecture of extracellular hemoglobin of Eophila tellinii

The molecular shape of the extracellular hemoglobin of the annelid worm Eophila tellinii was investigated by electron microscopy of negatively stained single molecules and of two-dimensional crystalline arrays. While the single molecules show the characteristic double hexagons, approx 28 nm in diameter and 19 nm in height, the molecules in the crystals are only 7–8 nm in height according to the 3D reconstruction. This is attributed to a dissociation of the hemoglobin complex; we present evidence that dissociation may proceed to the level of the main subunit from which half-molecules are reassembled. 3D reconstructions of two different crystal forms yield almost identical results and provide some information about the mass distribution within the main subunit. The presence or absence of the “central subunit” is tentatively interpreted in terms of a gross conformational change which entails a redistribution of mass also in the main subunit.     

Quaternary structure of the isolated restriction endonuclease EndoR.Bgl I from Bacillus globigii as revealed by electron microscopy.

The restriction endonuclease EndoR · BglI was purified nearly to homogeneity. BglI samples, when negatively stained with 4% uranyl acetate, show two different particle projections in the electron microscope. Projection A has an outer diameter of 22.5 ± 0.8 nm and is composed of six intensity maxima arranged in a ring; the centre of the ring exhibits slightly visible additional substructures. Projection B is also a ring; its outer diameter is 23.8 ± 0.7 nm; it does not show detailed fine structure, aside from a probable 10-fold rotational symmetry. Variations of the negative staining technique (single carbon layer, 2% uranyl acetate; ‘sandwich’ preparation with 4% uranyl acetate) revealed additional fine structural details for both projections. From the electron microscopic observations, a model of the enzyme particle was developed containing 20 identical, biologically active monomers of molecular weight around 61,000, arranged as a pentagonal dodecahedron. Tilting experiments established this structure decisively by interconversion of the different appearances of given particles in the expected way. By sodium dodecyl sulphate/polyacrylamide gel electrophoresis, polyacrylamide gel electrophoresis in a continuous molecular sieve gradient and evaluation of negatively stained enzyme particles, a molecular weight of the monomer of 61,000 was estimated, resulting in a total enzyme particle molecular weight of 1.2 × 10⁶ also determined by linear sucrose density-gradient centrifugation.     

Electron microscopy and image analysis of the multicatalytic proteinase

One electron micrographs, negatively stained multicatalytic proteinase molecules are viewed end-on (ring shaped) or side-on (rectangular shaped). For aurothioglucose, ammonium molybdate- and phosphotungstate-stained molecules, the dimensions measured are consistent. In contrast, uranyl acetate-staining reveals ring-shaped particles which vary in diameter between 12 and 16 nm. This is due to a partial collapse and substantial flattening of the structure. Digital image analysis of side-on views of the particles reveals a tripartite, reel-shaped structure. Within the ring-like, end-on projections of ammonium molybdate-stained molecules six local centres of mass can be discerned; their position appears to depart, however, from a true six-fold symmetry.     

Arrangement of Herpesvirus Deoxyribonucleic Acid in the Core

The core of the herpes simplex virion consists of an electron-dense toroidal structure 50 nm high, with an inside diameter of 18 nm and an outside diameter of 70 nm, penetrated by a less dense cylindrical mass. The toroid contains deoxyribonucleic acid (DNA), as evident from the observation that uranyl ion staining is removed by ethylenediaminetetraacetic acid under conditions which result in the extraction of uranyl ions bound to nuclear DNA. Studies on negatively stained preparations of purified capsids suggest that the toroid consists of DNA arranged as if it were spooled around the cylindrical mass.     

X-ray crystallographic and biochemical characterization of single crystals formed by proteolytically modified human fibrinogen

Large single crystals (0.7 mm X 0.4 mm X 0.3 mm) of human fibrinogen, modified with a crude exoprotease from Pseudomonas aeruginosa, have been obtained. The crystals are orthorhombic, space group P212121, with a = 9.5 +/- 0.1 nm, b = 11.1 +/- 0.1 nm, c = 44.0 +/- 0.4 nm. Their X-ray diffraction patterns extend to beyond 1.0 nm resolution. The asymmetric unit contains one fragment of 245 kDa molecular mass made up of an intact gamma chain, a slightly shortened beta chain and an N-terminal part (about one-third) of the alpha chain. In electron micrographs of rotary-shadowed samples the crystallized particles are very similar in size and shape to the well-known trinodular form of native fibrinogen. From the unit-cell dimensions and the intensity pattern a model is proposed in which the molecules consist of two halves related by a local twofold rotation axis, and are aligned with a displacement of multiples of 1/4 of their length giving a pseudohexagonal packing scheme.     

Electron Microscopic Study of Membranes and Walls of Bacteria and Changes Occurring During Growth Initiation

Thin sections of stationary-phase Streptococcus lactis cells showed that the wall and membrane are 20 and 7 nm thick, respectively. Whole cells were examined by negative staining with ammonium molybdate and by shadowing. On air-drying of whole cells, the membrane pulled away from the wall revealing adhesions between these organelles. Adhesions could not be seen after subculture of the stationary-phase cells into complex media or into solutions containing glucose, KCl, and CaCl(2) in tris(hydroxymethyl)aminomethane buffer. The adhesions were also observed in stationary-phase cells of other gram-positive bacteria. Fractured freeze-etched cells of S. lactis had a smooth outside surface, but the inside of the wall (or outside of the membrane) had a regular structure, repeating at 10 nm, which could correspond to the adhesions observed in the negatively stained air-dried cells. Freeze-etching also revealed holes in the outside wall which had the shape of inverted truncated cones. The outside diameter of the cone was 60 nm, and the diameter on the inside surface of the wall was 20 nm. The membrane had upstanding plugs, 20 nm in diameter, which could fill the holes in the wall.     

Negative staining studied by TEM and STEM mass measurements: Preliminary observations on collagen sheets negatively stained with uranyl acetate

Quantitative mass measurements by dark-field scanning transmission EM and conventional bright-field transmission EM have been used to determine the increase in mass brought about by negative staining. pN-collagen (which forms sheets of uniform thickness and known mass per unit area) was used as a test specimen; the negative stain was uranyl acetate (1%, pH 4.4). The mass increase corresponded to the addition of roughly 8 uranyl acetate molecules per nm² for lightly negatively stained specimens; for heavily stained specimens, it was 30 molecules or more. The appearance of the image was related to the mass increase. This preliminary study shows that mass measurements can provide a basis for the quantitative interpretation of images from negatively stained specimens.     

Nanoscale Probing Reveals that Reduced Stiffness of Clots from Fibrinogen Lacking 42 N-Terminal Bβ-Chain Residues Is Due to the Formation of Abnormal Oligomers

Removal of Bβl-42 from fibrinogen by Crotalus atrox venom results in a molecule lacking fibrinopeptide B and part of a thrombin binding site. We investigated the mechanism of polymerization of desBβ1-42 fibrin. Fibrinogen trinodular structure was clearly observed using high resolution noncontact atomic force microscopy. E-regions were smaller in desBβ1-42 than normal fibrinogen (1.2 nm ± 0.3 vs. 1.5 nm ± 0.2), whereas there were no differences between the D-regions (1.7 nm ± 0.4 vs. 1.7 nm ± 0.3). Polymerization rate for desBβ1-42 was slower than normal, resulting in clots with thinner fibers. Differences in oligomers were found, with predominantly lateral associations for desBβ1-42 and longitudinal associations for normal fibrin. Clot elasticity as measured by magnetic tweezers showed a G′ of ∼1 Pa for desBβ1-42 compared with ∼8 Pa for normal fibrin. Spring constants of early stage desBβ1-42 single fibers determined by atomic force microscopy were ∼3 times less than normal fibers of comparable dimensions and development. We conclude that Bβ1-42 plays an important role in fibrin oligomer formation. Absence of Bβ1-42 influences oligomer structure, affects the structure and properties of the final clot, and markedly reduces stiffness of the whole clot as well as individual fibrin fibers.