Gap junction structures. VIII. Membrane cross-sections.

Profiles of negatively stained gap junctions have been measured by grid sectioning. After normal levels of electron irradiation, the membrane thickness shrinks to about half that of unirradiated controls, but no shrinkage occurs in the hexagonal lattice plane. Even under low irradiation conditions, there is significant thinning of the membranes. Edge views, in which rows of connexons are aligned parallel to the beam, were obtained from grid sections, folds in normal negatively stained specimens, and sections of a positively stained specimen. Averaging these micrographs with the translational and mirror symmetry of the projected lattice image displays conserved and variable features in the stain distribution of different specimens. Variations in the relative amount of negative stain in the gap at the surfaces and in the channel are uncorrelated with the irradiation but appear to depend on the local staining conditions and the integrity of the connexons. The dimensions measured from previously unirradiated grid sections, folds, and positively stained sections are in accord with x-ray diffraction measurements. Radiation-induced shrinkage can be accounted for by mass loss principally from the membrane bilayer. Disordering of the surface structure appears to be correlated with the radiation sensitivity of the bilayer; in contrast, the gap structure is well preserved under a variety of conditions.     

Correlation analysis of gap junction lattice images.

Fourier averages of connexon images computed from low-irradiation electron micrographs of isolated negatively stained gap junction domains exhibited differences in stain distribution and connexon orientation. To analyze these polymorphic structures, correlation averaging methods were applied to images from negatively stained and frozen-hydrated specimens. For the negatively stained specimens, separate averages over two subsets of connexons with differing degrees of stain accumulation in the axial channel were obtained. Two populations of connexons with opposite skew orientations were distinguishable within a single junctional domain of a frozen-hydrated specimen. Correlation maps calculated using the left- and right-skewed references showed that the selected connexons tend to locally cluster. Using correlation methods to analyze packing disorder in a typical connexon lattice, we estimated the root-mean-square variation in the nearest neighbor pair separation to be approximately 11% of the lattice constant. Displacements of the connexons relative to each other increased with increasing pair separation in the lattice, rather like a liquid, although long-range orientation order was conserved as in a crystal. These results support the hypothesis that the hexagonal ordering of the connexons results from short-range repulsive forces.     

A survey of 50 S ribosomal subunits by dark field electron microscopy

A survey of dark field electron micrographs of the 50 S ribosomal subunit of E. coli has been performed and supplemented, for comparative purposes, by examination of negatively stained or metal shadowed specimens in the bright field mode. Attention was directed to the so-called "crown" and "kidney" views. The elongated appendage seen in negatively stained crown profiles was not observed in unstained or positively stained samples examined in dark field; these showed only symmetrical crown profiles regardless of changes in buffer type and drying method and of the presence or absence of uranyl acetate treatment and glutaraldehyde fixation. The crown view occasionally displayed a bifurcation in one of the lateral lobes, while the kidney profile showed a groove near the base of the convex edge. Uranyl acetate treatment produced delicate stripes which may give an indication of the surface RNA distribution.     

The pleated sheet: an unusual negative-staining method for transmission electron microscopy of biological macromolecules

A novel method for preparing negatively stained specimens is described which appears to improve the routine resolution of biological structure in direct images obtained by transmission electron microscopy. In the new method, which we term the pleated sheet technique, macromolecules are adsorbed to a carbon film by the Valentine procedure (R. Valentine, B. Shapiro, and E. Stadtman (1968) Biochemistry, 7, 2143-2152), and the film then carefully pleated while in contact with a 1% uranyl formate solution to trap stain within the folds of pleats. A grid is placed on the compressed film, and film plus grid retrieved with a Saran Wrap drum. Subsequent dehydration produces a filmed grid containing negatively stained macromolecules within the folds of pleated regions and positively stained macromolecules in single sheet regions. The effect of sandwiching sample and stain between carbon layers is to produce exceedingly uniform negative staining so that stain contours more accurately and more reproducibly reflect true molecular contours. Electron micrographs of IgG and IgA molecules prepared by these methods are exhibited that permit unambiguous comparison of structure imaged in the electron microscope against known structures solved by single-crystal X-ray diffraction. Correlation is excellent; the smallest resolvable element in micrographs is an immunoglobulin domain, whose molecular weight is 12 000.     

Gap junction structures. VII. Analysis of connexon images obtained with cationic and anionic negative stains

Micrographs of isolated gap junction specimens, negatively stained with one molybdate, three tungstate and three uranyl stains, were recorded at low and high irradiation. Fourier-averaged images of the negatively stained gap junctions have been self-consistently scaled to identify conserved and variable features. Intrinsic features in the hexagonally averaged images have been distinguished from residual noise by statistical comparisons among similarly prepared specimens. The cationic uranyl stains can penetrate the axial connexon channel, whereas the anionic stains are largely excluded; these observations indicate that the channel is negatively charged. Variability in the extent of the axial stain penetration, and enhancement of this staining by radiation damage and heating may be accounted for by a leaky, labile channel gate. The peripheral stain concentrations marking the perimeter of the skewed, six-lobed connexon image and the stain-excluding region at the 3-fold axis of the lattice, which are seen only under conditions of low irradiation with both anionic and cationic stains, are identified as intrinsic features of the isolated gap junction structure. The stain concentrations located approximately 30 A from the connexon center appear to be symmetrically related on opposite sides of the junction by non-crystallographic 2-fold axes oriented approximately 8 degrees to the lattice axes at the plane of the gap. The radiation-sensitive hexagonal features seen in the negatively stained images may correspond to substructure on the cytoplasmic surfaces of the paired gap junction membranes.     

Multivariate statistical analysis of ribosome electron micrographs. L and R lateral views of the 40 S subunit from HeLa cells

The morphology of the small (40 S) subunit of the eukaryotic ribosome from HeLa cells has been examined by single-particle averaging and multivariate image analysis applied to electron micrographs of negatively stained specimens. The use of multivariate image analysis allows different, independent components of the structural variation within the particles to be identified and separately studied. The largest component of variance for both lateral views (termed L and R) was the variation in the peripheral stain intensity. The second largest component of interparticle variation is due to changes in the particle appearance most likely associated with a change of orientation on the specimen film.Averages formed from particles falling within a small range of peripheral stain intensity allowed the changes in the projected structure to be studied as a function of local stain level. Visual observations of stain variation could be confirmed quantitatively.Significant differences were found between averages of particles in the L view and those in the R view. Multivariate image analysis of a mixed population of L and mirrored R particles showed that the differences consistently affect all particles. However, the R view increasingly resembles the L view as the overall level of stain is increased, in agreement with a model of partial stain immersion.     

Cryo-EM of the native structure of the calcium release channel/ryanodine receptor from sarcoplasmic reticulum.

The native structure of the calcium release channel (ryanodine receptor) from rabbit skeletal muscle has been analyzed in two dimensions from electron micrographs of frozen hydrated specimens. Within a resolution of 3.0 nm there is excellent agreement between the structure as seen in vitreous water and in negative stained specimens. Features seen in the three-dimensional reconstruction of the negatively stained channel can be identified in the projection of the unstained receptor.     

Electron microscopy and computer image averaging of ice-embedded large ribosomal subunits from Escherichia coli

Electron micrographs of frozen-hydrated, large ribosomal subunits from Escherichia coli have been analyzed by computer image processing. Images of subunits in the so-called "crown" orientation were analyzed by correlation alignment procedures developed for negatively stained specimens. Averages of the aligned images showed both similarities and differences to averages determined for negatively stained specimens. The L1 ridge is more dense and stalk-like in frozen-hydrated as compared with negatively stained subunits, possibly because it is associated with ribosomal RNA. The results show that it should be feasible to determine the three-dimensional structure of the large ribosomal subunit from micrographs of individual, frozen-hydrated subunits that have been tilted in the electron microscope.     

Gap junction structures. IV. Asymmetric features revealed by low- irradiation microscopy

Micrographs of mouse liver gap junctions, isolated with detergents, and negatively stained with uranyl acetate, have been recorded by low-irradiation methods. Our Fourier-averaged micrographs of the hexagonal junction lattice show skewed, hexameric connexons with less stain at the threefold axis than at the six indentations between the lobes of the connexon image. These substructural features, not clearly observed previously, are acutely sensitive to irradiation. After an electron dose less than that normally used in microscopy, the image is converted to the familiar doughnut shape, with a darkly stained center and a smooth hexagonal outline, oriented with mirror symmetry in the lattice. Differences in appearance among 25 reconstructed images from our low-irradiation micrographs illustrate variation in staining of the connexon channel and the space between connexons. Consistently observed stain concentration at six symmetrically related sites approximately 34 A from the connexon center, 8 degrees to the right or left of the (1, 1) lattice vector may reveal an intrinsic asymmetric feature of the junction structure. The unexpected skewing of the six-lobed connexon image suggests that the pair of hexagonal membrane arrays that form the junction may not be structurally identical. Because the projected image of the connexon pair itself appears mirror symmetric, each pair may consist of two identical connexon hexamers related by local (noncrystallographic) twofold axes in the junctional plane at the middle of the gap. All connexons may be chemically identical, but their packing in the hexagonal arrays on the two sides of the junction appears to be nonequivalent.     

The development and application of negative staining to the study of isolated virus particles and their components: A personal account

The subsequent development and application of the negative staining technique to isolated virus particles and their components, following the early studies on the structure of T2 bacteriophage proteins, is described. The use of the method to prepare particles covering a wide range of different types of virus for examination in the electron microscope is reviewed, together with more recent advances in image analysis of electron micrographs obtained from negatively stained virus specimens.     

Comparison of the decameric structure of peroxiredoxin-II by transmission electron microscopy and X-ray crystallography.

The decameric human erythrocyte protein torin is identical to the thiol-specific antioxidant protein-II (TSA-II), also termed peroxiredoxin-II (Prx-II). Single particle analysis from electron micrographs of Prx-II molecules homogeneously orientated across holes in the presence of a thin film of ammonium molybdate and trehalose has facilitated the production of a >/=20 A 3-D reconstruction by angular reconstitution that emphasises the D5 symmetry of the ring-like decamer. The X-ray structure for Prx-II was fitted into the transmission electron microscopic reconstruction by molecular replacement. The surface-rendered transmission electron microscopy (TEM) reconstruction correlates well with the solvent-excluded surface of the X-ray structure of the Prx-II molecule. This provides confirmation that transmission electron microscopy of negatively stained specimens, despite limited resolution, has the potential to reveal a valid representation of surface features of protein molecules. 2-D crystallisation of the Prx-II protein on mica as part of a TEM study resulted in the formation of a p2 crystal form with parallel linear arrays of stacked rings. This latter 2-D form correlates well with that observed from the 2.7 A X-ray structure of Prx-II solved from a new orthorhombic 3-D crystal form.     

Biochemical and ultrastructural characterization of a high molecular weight soluble Mg2+ -ATPase from human erythrocytes

The isolation and purification of a 600,000 Mr cytosolic Mg2+ -ATPase from human erythrocytes is described. The electrophoretic properties of the native and sodium dodecyl sulphate-dissociated protein are presented and compared with those of the erythrocyte protein cylindrin . The Mg2+-ATPase has a single subunit of Mr 100,000 and it has an isoelectric point of 4.9. From transmission electron microscopy of negatively stained specimens, it is proposed that the Mg2+-ATPase is hexameric, containing two superimposed trimers of the 100,000 Mr subunit, which gives rise to a 13 nm pseudohexagonal particle with a central 3 nm cavity. Varying the orientation of the protein in the negative stain also produces images that are not hexagonal. When orientated on-edge, the protein produces a double-disc image, which is most clearly defined under acidic negative staining conditions with uranyl acetate, when some aggregation of the protein is produced. The ultrastructure of the Mg2+-ATPase is shown to be distinctly different from that of cylindrin . A comparative discussion of the negatively stained transmission electron microscopical images of the Mg2+-ATPase, mitochondrial F1-ATPase and several other oligomeric proteins and enzymes is presented.     

Membrane attach complex of complement (MAC): three-dimensional analysis of MAC-phospholipid vesicle recombinants

The three-dimensional structure of recombinants of the isolated membrane attack complex (MAC) of complement with single bilayer dioleoyllecithin (DOL) vesicles and with dimyristoyllecithin (DML) vesicles was determined. A total of four MAC-vesicle complexes were analyzed by imaging negatively stained specimens at various defined tilting angles under minimal dose conditions in the electron microscope and by computer-aided three-dimensional reconstruction. The information on electron micrographs obtained at 6 degrees angular increments from +60 degrees to -60 degrees was digitized by densitometric scanning, Fourier-transformed, corrected for imaging errors, cross-correlated, and synthesized to the three-dimensional image. All four MAC-vesicle recombinants showed stain penetration into the interior of the vesicle, indicating increased permeability of the bilayer to negative stain. The MAC appeared as a hollow structure of 16-nm height, 2.0-nm wall thickness, and a 3.0-nm torus at the free end with an outer and inner diameter of 20.0 nm and 10.0 nm. In MAC-DOL vesicles the hollow core of the MAC terminated at the membrane-binding site, and only small pores of up to 2.0-nm in diameter penetrated the bilayer. In one MAC-DML vesicle lipid discontinuities on the outer circumference of the MAC binding site mediated stain penetration. The second MAC-DML vesicle showed a channel of approximately 4.0 nm connecting the hollow core of the MAC across the bilayer with the vesicle interior. The results suggest the MAC may mediate increased membrane permeability by protein channel formation in addition to lipid reorientation.     

Electron microscopic studies of negatively stained liposomes and membrane intrinsic polypeptide

High resolution electron microscopy has been applied to the study of the permeability properties of liposomes reconstituted with lens fibre protein. The experiments showed that liposomes formed in the presence of main intrinsic polypeptide (MIP) of bovine lens fibres appeared to become modified by changes in shape; also the bilayers seen in the electron micrographs of negatively stained specimens showed evidence of some structural changes or granularity. The morphological appearance of liposomes formed in the presence of MIP were interpreted as being consistent with the results of experiments performed by other methods.     

Electron microscopy of bacteriophage phi 6 nucleocapsid: two-dimensional image analysis.

Electron micrographs of negatively stained nucleocapsids isolated from intact, wild-type phi 6 bacteriophage revealed three distinct morphological forms. Two-dimensional analysis of electron micrographs of two of these forms and image averaging of all forms are consistent with a dodecahedral structure embodied in the phi 6 nucleocapsid.     

Three-dimensional structure of the large ribosomal subunit from Escherichia coli.

The three-dimensional structure of the large (50S) ribosomal subunit from Escherichia coli has been determined from electron micrographs of negatively stained specimens. A new method of three-dimensional reconstruction was used which combines many images of individual subunits recorded at a single high tilt angle. A prominent feature of the reconstruction is a large groove on the side of the subunit that interacts with the small ribosomal subunit. This feature is probably of functional significance as it includes the regions where the peptidyl transferase site and the binding locations of the elongation factors have been mapped previously by immunoelectron microscopy.     

Helical structure of Bordetella pertussis fimbriae.

The helical structures of Bordetella pertussis fimbriae of serotypes 2 and 6 were determined by optical diffraction analysis of electron micrographs of negatively stained paracrystalline bundles of purified fimbriae. The fimbrial structure is based on an axial repeat of 13 nm that contains five repeating units in two complete turns of a single-start helix. This structure was confirmed by direct measurements of mass per unit length for individual fimbriae performed by dark-field scanning transmission electron microscopy of unstained specimens. These data further established that the helically repeating unit is a monomer of fimbrial protein (Mr congruent to 22,000 for type 2 and Mr congruent to 21,500 for type 6). Radial density profiles calculated from the scanning transmission electron micrographs showed that the fimbria has peak density at its center, i.e., no axial channel, consistent with the results of conventional negative-staining electron microscopy. The radial profile gives an outermost diameter of approximately 7.5 nm, although the peripheral density is, on average, diffuse, allowing sufficient intercalation between adjacent fimbriae to give a center-to-center spacing of approximately 5.5 nm in the paracrystals. Despite serological and biochemical differences between type 2 and type 6 fimbriae, the packing arrangements of their fimbrial subunits are identical. From this observation, we infer that the respective subunits may have in common conserved regions whose packing dictates the helical geometry of the fimbria. It is plausible that a similar mechanism may underlie the phenomenon of phase variations in other systems of bacterial fimbriae.     

Model for the capsid structure of alfalfa mosaic virus.

The coat protein of alfalfa mosaic virus is clustered in such a way as to avoid the 3 and 6-fold lattice positions of the hexagonal surface lattice. This results in a relatively open structure for the capsid, which is mainly caused by holes situated at the 6-fold positions and, to a minor extent, those present at the 3-fold lattice points. The evidence for this has been obtained by analysing electron micrographs of negatively stained virus particles by optical diffraction and digital image processing.     

Three-dimensional image analysis of the complex of thin filaments and myosin molecules from skeletal muscle. I. Tilt angle of myosin subfragment-1 in the rigor complex

A three-dimensional image of the "rigor" complex of actin and chymotryptic myosin subfragment-1 was reconstituted from electron micrographs of negatively stained specimens. Data went out to 20 A radially and 26 A axially. The reconstituted images allowed us to deduce the angle between the major axis of the main part of myosin subfragment-1 and the axis of the actin helix. The subfragment-1 molecules were attached to the actin filament in a configuration in which they were tilted by only about 15 degrees from the plane perpendicular to the axis of the actin helix. The implication of the smaller tilt angle than the commonly accepted value is discussed.     

Crystals of Lumbricus erythrocruorin

Lumbricus terrestris erythrocruorin, a 3.9 X 10(6) Mr respiratory protein, has been crystallized in four different forms. Despite the high molecular symmetry apparent from images in electron micrographs, only one crystal form expresses any molecular symmetry as crystallographic symmetry. The lattice parameters provide upper limits on the molecular dimensions of 267 A X 308 A X 172 A (1 A = 0.1 nm), which agree well with dimensions obtained from electron micrographs of negatively stained molecules. We have collected diffraction data to 5.5 A from type III crystals and have begun a structural analysis.