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.     

Electron microscopy of the stacked disk aggregate of tobacco mosaic virus protein. II. The influence of electron irradiation of the stain distribution

Optical and computer analysis of bright field electron micrographs of the same negatively stained stacked disk specimens subjected to different electron doses shows that the stain migrates and redistributes itself over the protein surfaces during irradiation. The analysis suggests that the main factors responsible for this migration are the contraction of the stain inevitably brought about by the irradiation, and associated surface energy effects. The morphology of the protein has a strong influence on the path of the migration.A major consequence of these changes in stain distribution is that conventional bright field micrographs taken under normal dosage conditions tend to give a somewhat misleading representation of the specimen's original structure.Migration of the stain under the electron beam is likely to be a fairly general phenomenon and it therefore seems important to take bright field micrographs of negatively stained specimens using the smallest possible electron doses.     

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.     

The grid sectioning technique: a study of catalase platelets.

The grid sectioning technique has been used to obtain the two missing principal axis projections of orthorhombic catalase platelets and to measure directly the unit cell c-value. The negatively stained platelets have a unit cell c-dimension of half that proposed by Unwin (1975) from powder X-ray diffraction. The precision of the grid sectioning technique in positioning sections along a specimen axis shows that the growth fault lines usually observed on negatively stained catalase platelets are rows of missing molecules filled with stain. From these sections conclusions are drawn concerning the action of negative stain on a specimen, the microtomy process, and the specimen/supporting film interaction. Finally the value of microtomy for detailed structural analysis of biological objects is emphasized.     

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.     

Structure of molluscan thick filaments: a common origin for diverse appearances.

Thick filaments from the smooth adductor muscles of the oysters Ostrea edulis and Crassostrea angulata have been examined in the electron microscope after negative staining. The two well-known patterns of stain (whose origin and relation have been uncertain), one a series of transverse narrow lines at intervals of 144 Å along the filament axis and the other a regular two-dimensional arrangement of stained spots (Bear &; Selby, 1956), are found to be mutually interconvertible by rotating the grid around the filament axis. This is interpreted to mean that the spots are the projections of stained regions running through the filament in a common direction. Only when looking along this direction will the net pattern be seen with maximum clarity and sharpness. On rotation of the filament round its axis, the spots broaden transversely to the axis, overlapping and ultimately only the axial periodicity will remain. The structure is therefore not helical, but resembles a crystal lattice, although no period can be discerned normal to the net plane.The addition of 10 mm-EDTA to all solutions used in the filament preparation (except the stain), especially when ammonium molybdate is the stain employed, removes many puzzling appearances (probably caused by positive staining) which render the interpretation difficult. The appearance of the negatively stained filament can be related to the stain patterns in negatively stained paramyosin paracrystals (Cohen et al., 1971).     

Image processing of electron micrographs of human alpha 2-macroglobulin half-molecules induced by Cd2+

Human alpha 2-macroglobulin is a tetrameric plasma inhibitor of proteinases. Its dissociation by Cd2+ gives functional dimers. Electron microscopy of negatively stained dimers shows their round-ended cylindrical shape with furrows delimiting 3 main stain-excluding domains. Image processing of electron micrographs shows the existence of 2 main orientations of the dimers on the carbon support film. The dimer is composed of 2 curved monomers linked in a central domain, and related by a 90 degree rotation. Taking into account the known primary structure of alpha 2-macroglobulin and the linkage of the 2 constitutive monomers by 2 disulfide bonds, the molecular organization of the dimer is discussed, extended to the tetrameric molecule and compared to the published models of human alpha 2-macroglobulin.     

Use of negative stain and single-particle image processing to explore dynamic properties of flexible macromolecules

Flexible macromolecules pose special difficulties for structure determination by crystallography or NMR. Progress can be made by electron microscopy, but electron cryo-microscopy of unstained, hydrated specimens is limited to larger macromolecules because of the inherently low signal-to-noise ratio. For three-dimensional structure determination, the single particles must be invariant in structure. Here, we describe how we have used negative staining and single-particle image processing techniques to explore the structure and flexibility of single molecules of two motor proteins: myosin and dynein. Critical for the success of negative staining is a hydrophilic, thin carbon film, because it produces a low noise background around each molecule, and stabilises the molecule against damage by the stain. The strategy adopted for single-particle image processing exploits the flexibility available within the SPIDER software suite. We illustrate the benefits of successive rounds of image alignment and classification, and the use of whole molecule averages and movies to analyse and display both structure and flexibility within the dynein motor.     

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).     

Structure and properties of novel inclusions in Shewanella putrefaciens

Cytoplasmic inclusions surrounded by a bilayer membrane were seen in thin sections. negatively stained and freeze-fractured preparations of Shewanella putrefaciens. Cells harvested from the late exponential and early stationary phase showed a higher number of these vesicles than bacteria isolated from early exponential or late stationary phase. Chemical dyes for polyphosphate or poly-beta-hydroxybutyrate did not stain the material enclosed within these vesicles. Elemental analysis of the material indicated that the content was organic in nature and might be a protein. HPLC analysis of the material showed that it was probably not a carbon source, nor an electron acceptor used by S. putrefaciens.     

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.     

Packing of ribosomes in crystals from the lizard Lacerta sicula

The packing of ribosomes in the large crystalline sheets found in the lizard Lacerta sicula has been investigated by electron microscopy. The ribosomes in each of the two layers composing a sheet are organised as tetramers on a P4 space group lattice. The two layers face in opposite directions and tend to be related to one another crystallographically, generating a family of P422 crystals of different unit cell dimensions. The projected structure of one layer was determined from negatively stained, isolated sheets by separating the contributions from each layer in Fourier transforms computed from electron micrographs. Comparison of the projection map with other, low resolution, analyses of images of isolated eukaryotic ribosomes indicates that the large subunit- small subunit axis lies approximately parallel to the plane of the sheet.     

Molecular shape of Lumbricus terrestris erythrocruorin studied by electron microscopy and image analysis

The molecular structure of erythrocruorin (hemoglobin) from Lumbricus terrestris has been studied by electron microscopy of negatively stained particles. Over 1000 molecular projections were selected from a number of electron micrographs and were then classified by multivariate statistical image-processing techniques. The two main groups of top and side views were each subdivided into smaller classes with significantly different features. About half of the top-view projections exhibit perfect hexagonal symmetry at the current resolution of about 2.0 nm, while the other top views lack this symmetry, probably as a result of tilting of the molecules relative to the carbon support film. The side views were separated into two 'families', each associated with the two different stable side-view positions the molecules can take. From these narrow stable side-views, the two families of projections are, again, generated by tilting. The symmetry properties of the three non-tilted projections show that Lumbricus erythrocruorin has a pointgroup D6 (622) symmetry rather than D3 (32).     

Three-dimensional structure of the mammalian cytoplasmic ribosome

A three-dimensional reconstruction of the 80 S ribosome from rabbit reticulocytes has been calculated from low-dose electron micrographs of a negatively stained single-particle specimen. At 37 A resolution, the precise orientations of the 40 S and 60 S subunits within the monosome can be discerned. The translational domain centered on the upper portion of the subunit/subunit interface is quite open, allowing considerable space between the subunits for interactions with the non-ribosomal macromolecules involved in protein synthesis. Further, the cytosolic side of the monosome is strikingly more open than the membrane-attachment side, suggesting a greater ease of communication with the cytoplasm, which would facilitate the inwards and outwards diffusion of a number of ligands. Although the 60 S subunit portion of the 80 S structure shows essentially all of the major morphological features identified for the eubacterial 50 S large subunit, it appears to possess a region of additional mass that evidently accounts for the more ellipsoidal form of the eukaryotic subunit.     

An X-ray Scattering Study of Bromegrass Mosaic Virus

X-ray scattering data and electron microscope observations are presented for bromegrass mosaic virus. Its radial density distribution is obtained from the Fourier transform of the amplitudes of the scattered x-rays. The results indicate that the virus is 260 A in diameter, it has an almost empty central cavity which is about 80 A in diameter, and the regions occupied by RNA and protein are approximately equal in average density. Electron micrographs of negatively stained preparations also give an outside diameter of 260 A and indicate that there is a central region about 90 A in diameter into which uranyl acetate can penetrate. Positively stained preparations indicate that the nucleic acid is concentrated in a shell-shaped region which is in turn surrounded by a shell of protein. In order for the RNA and protein regions to have the same average electron density the RNA must have a hydration of 1.29 gm of water per gm of RNA and the protein must have a hydration of 0.24 gm of water per gm of protein.     

Molecular organization in molluscan operculae.

Operculin is a glycine-rich protein present as the major component of gastropod operculae. X-ray and infrared studies of operculins lead to the conclusion that operculae contain antiparallel-chain pleated sheets oriented so that the plane of the pleated sheet is parallel to the plane of the operculum, which is a flat ovoidal or circular sheet. Partial hydrolysis gives evidence of repeating Asp-Gly-Asp and Asp-Ala-Asp sequences as well as of regions rich in Gly.     

On the freezing and identification of lipid monolayer 2-D arrays for cryoelectron microscopy

Lipid monolayers provide a convenient vehicle for the crystallization of biological macromolecules for 3-D electron microscopy. Although numerous examples of 3-D images from 2-D protein arrays have been described from negatively stained specimens, only six structures have been done from frozen-hydrated specimens. We describe here a method that makes high quality frozen-hydrated specimens of lipid monolayer arrays for cryoelectron microscopy. The method uses holey carbon films with patterned holes for monolayer recovery, blotting and plunge freezing to produce thin aqueous films which cover >90% of the available grid area. With this method, even specimens with relatively infrequent crystals can be screened using automated data collection techniques. Though developed for microscopic examination of 2-D arrays, the method may have wider application to the preparation of single particle specimens for 3-D image reconstruction.     

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural biology ¹. In recent years, this method has achieved great success in studying structures of proteins and macromolecular complexes ^{2, 3}. Compared with electron cryomicroscopy (cryoEM), in which frozen hydrated protein samples are embedded in a thin layer of vitreous ice ⁴, negative staining is a simpler sample preparation method in which protein samples are embedded in a thin layer of dried heavy metal salt to increase specimen contrast ⁵. The enhanced contrast of negative stain EM allows examination of relatively small biological samples. In addition to determining three-dimensional (3D) structure of purified proteins or protein complexes ⁶, this method can be used for much broader purposes. For example, negative stain EM can be easily used to visualize purified protein samples, obtaining information such as homogeneity/heterogeneity of the sample, formation of protein complexes or large assemblies, or simply to evaluate the quality of a protein preparation.In this video article, we present a complete protocol for using an EM to observe negatively stained protein sample, from preparing carbon coated grids for negative stain EM to acquiring images of negatively stained sample in an electron microscope operated at 120kV accelerating voltage. These protocols have been used in our laboratory routinely and can be easily followed by novice users.     

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.     

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.