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.     

Ribosome structure: hand determination by electron microscopy of 30 S subunits.

Negatively stained Escherichia coli 30 S ribosome subunits have been examined by electron microscopy at tilt angles of ?45 °, 0 ° and +45 °. The tilted views, together with images of metal shadowed particles are consistent with an asymmetric model for the structure of the 30 S subunit and enable the absolute hand of the particle to be determined.     

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.     

Fine Structures of Cell Envelopes of Chlamydia Organisms as Revealed by Freeze-Etching and Negative Staining Techniques

The cell walls of Chlamydia psittaci (meningopneumonitis strain) were examined by the freeze-etching and negative staining techniques. It was observed that the cleaved convex surface of the developmental, reticulate body was covered with numerous non-etchable particles 9 to 10 nm in diameter, these particles being rarely seen on the concave surface. Similarly, the convex surface of the mature, elementary body (EB) was covered with many particles but the concavity lacked these particles. After etching, the smooth concave surface of the EB appeared to have a hexagonally arrayed subunit structure, on which the button structure (B structure) was observed. Each B structure had a diameter of 27 nm and several B structures were grouped together in a hexagonal arrangement with a center-to-center spacing of 45 nm. In a limited area of the negatively stained EB cell wall, hexagonally arrayed rosette structures were present, with a center-to-center spacing similar to the B structures seen in the freeze-etched preparation. Each rosette, about 19 to 20 nm in diameter, appeared to be composed of a radial arrangement of nine subunits. The freeze-fractured cell wall-cytoplasmic membrane complexes indicated that the outer surface of the cytoplasmic membrane which appeared as the convex surface was covered with the fine particles, and thus it was likely that frozen EB was cleaved at the gap between the cell wall and ctyoplasmic membrane. On the cleaved inclusion, several groups of fine particles were observed. In each group, the particles were arranged hexagonally with the spacing ranging from 20 to 50 nm.     

Three-dimensional reconstruction of the valyl-tRNA synthetase/elongation factor-1H complex and localization of the delta subunit

Eukaryotic valyl-tRNA synthetase (ValRS) and the heavy form of elongation factor 1 (EF-1H) are isolated as a stable high molecular mass complex that catalyzes consecutive steps in protein biosynthesis--aminoacylation of tRNA and its transfer to elongation factor. Herein is the first three-dimensional structure of the particle as calculated from electron microscopic images of negatively stained samples of the human ValRS/EF-1H complex. The ca. 12 x 8 nm particle has two distinct domains and each appears to have twofold symmetry. Bound antibodies place two delta subunits near the particle's center. These data support a dimeric head-to-head arrangement of particle components.     

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.     

Electron microscopic analysis of the peripheral and membrane parts of mitochondrial NADH dehydrogenase (complex I).

Two related forms of the respiratory chain NADH dehydrogenase (NADH:ubiquinone reductase or complex I) are synthesized in the mitochondria of Neurospora crassa. Normally growing cells make a large form that consists of 25 subunits encoded by nuclear DNA and six to seven subunits encoded by mitochondrial DNA. Cells grown in the presence of chloramphenicol, however, make a smaller form comprising only 13 subunits, all encoded by nuclear DNA. When the large enzyme is dissected by chaotropic agents (such as NaBr), all those subunits of the large form that are missing in the small form can be isolated as a distinct, so-called hydrophobic fragment. The small enzyme and the hydrophobic fragment make up, with regard to their redox groups, subunit composition and function, two complementary parts of the large-form NADH dehydrogenase. Averaging of electron microscope images of single particles of the large enzyme was carried out, revealing an unusual L-shaped structure with two domains or "arms" arranged at right angles. The hydrophobic fragment obtained by the NaBr treatment corresponds in size and appearance to one of these arms. A three-dimensional reconstruction from images of negatively stained membrane crystals of the large-form NADH dehydrogenase shows a peripheral domain, protruding from the membrane, with weak unresolved density within the membrane. This peripheral domain was removed by washing the crystals in situ with 2 M-NaBr, exposing a large membrane-buried domain, which was reconstructed in three dimensions. A three-dimensional reconstruction of the small enzyme from negatively stained membrane crystals, also described here, shows only a peripheral domain. These results suggest that the membrane protruding arm of the large form corresponds to the small enzyme, whereas the arm lying within the membrane can be identified as the hydrophobic fragment. The two parts of NADH dehydrogenase that can be defined by the separate genetic origin of (most of) their subunits, their independent assembly, and their distinct contributions to the electron pathway can thus be assigned to the two arms of the L-shaped complex I.     

Characterization by electron microscopy of isolated particles and two-dimensional crystals of the CP47-D1-D2-cytochrome b-559 complex of photosystem II

A photosystem II complex containing the reaction center proteins D1 and D2, a 47-kDa chlorophyll-binding protein (CP47), and cytochrome b-559 was isolated with high yield, purity, and homogeneity; small but well-ordered two-dimensional crystals were prepared from the particles. The crystals and the isolated particles were analyzed by electron microscopy using negatively stained specimens. The information of 20 different digitized crystals was combined by alignment programs based on correlation methods to obtain a final average. The calculated diffraction pattern, with spots up to a resolution of 2.5 nm, and the optical diffraction pattern of a single crystal indicate that the plane group is p22121 (also called p2gg) and that the unit cell is rectangular with parameters of 23.5 x 16.0 nm, containing four stain-excluding monomers (two face-up and two face-down). In projection, the monomers have an asymmetrical shape with a length of 10 nm, a maximal width of 7.5 nm, and a height of 6 nm; their molecular mass is 175 +/- 40 kDa.     

Amino-acid-transport mutant of Nicotiana tabacum L.

The structure of spinach ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) has been investigated by tilted-view electron microscopy of negatively stained monolayer crystals and image processing. The structure determined consists of a cylinder of octagonal cross-section with a large central hole. Based on this and other available evidence a model for the arrangement of the large and small subunits is suggested with the eight small subunits arranged equatorially around the core of eight large subunits.Abbreviations LS large subunit - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - SS small subunit     

Trinodular structure of fibrinogen. Confirmation by both shadowing and negative stain electron microscopy

Fibrinogen molecules sprayed on mica, dried in vacuo and shadowed with platinum appear as trinodular rods. We describe here the flotation technique of negative staining by which we were able to visualize individual fibrinogen molecules. The fibrinogen molecules in our negatively stained preparations have a trinodular structure identical to that of shadowed molecules. We believe that the 20 nm diameter globules seen in previous negative staining studies are aggregates of these molecules. This is the first study in which both shadowed and negatively stained preparations of fibrinogen support a single, consistent model for the structure of fibrinogen. The molecules in our preparation are 45 nm long (±2.5 nm, our overall estimate of accuracy), the two outer nodules are 6 to 7 nm in diameter and the middle nodule is 4 to 5 nm in diameter.     

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.     

The reaction center complex from the green sulfur bacterium Chlorobium tepidum: a structural analysis by scanning transmission electron microscopy.

The three-dimensional (3D) structure of the reaction center (RC) complex isolated from the green sulfur bacterium Chlorobium tepidum was determined from projections of negatively stained preparations by angular reconstitution. The purified complex contained the PscA, PscC, PscB, PscD subunits and the Fenna-Matthews-Olson (FMO) protein. Its mass was found to be 454 kDa by scanning transmission electron microscopy (STEM), indicating the presence of two copies of the PscA subunit, one copy of the PscB and PscD subunits, three FMO proteins and at least one copy of the PscC subunit. An additional mass peak at 183 kDa suggested that FMO trimers copurify with the RC complexes. Images of negatively stained RC complexes were recorded by STEM and aligned and classified by multivariate statistical analysis. Averages of the major classes indicated that different morphologies of the elongated particles (length=19 nm, width=8 nm) resulted from a rotation around the long axis. The 3D map reconstructed from these projections allowed visualization of the RC complex associated with one FMO trimer. A second FMO trimer could be correspondingly accommodated to yield a symmetric complex, a structure observed in a small number of side views and proposed to be the intact form of the RC complex.     

Three-dimensional reconstruction of innermost chorion layer from Drosophila melanogaster

A low-resolution three-dimensional structure of the crystalline innermost chorion layer (ICL) has been calculated from electron microscope images of tilted negatively stained crystals. The isolated ICL is a single layer, about 12 nm thick and appears to be made up of two types of subunits, each approximately 3 nm in diameter, arranged regularly as groups of four heterodimers in space group C222. Linking density between these groups of subunits, maintaining the integrity of the layer, appears to be confined mainly to the outer surfaces of the ICL.     

Three-dimensional reconstruction of innermost chorion layer from Drosophila melanogaster

A low-resolution three-dimensional structure of the crystalline innermost chorion layer (ICL) has been calculated from electron microscope images of tilted negatively stained crystals. The isolated ICL is a single layer, about 12 nm thick and appears to be made up of two types of subunits, each approximately 3 nm in diameter, arranged regularly as groups of four heterodimers in space group C222. Linking density between these groups of subunits, maintaining the integrity of the layer, appears to be confined mainly to the outer surfaces of the ICL.     

Subunit structure of paired helical filaments in Alzheimer''s disease

The neurofibrillary tangles that occur in the brain in cases of senile dementia of the Alzheimer type contain a distinctive type of filament, the paired helical filament (PHF). We have developed a method for isolating the tangles postmortem in sufficient yield for structural study of PHFs by electron microscopy of negatively stained and shadowed preparations. This material shows the characteristic helical structure seen in sectioned embedded material. In addition, two striking fragmentation patterns are observed. (a) Some filaments show sharp transverse breaks at apparently random positions along the filament. (b) In a few PHFs one strand is missing for a variable length, whereas the other appears to maintain its structural integrity. The shadowed specimens show the PHF to be wound in a left-handed manner. These observations indicate that the PHF consists of subunits of very limited axial extent arranged along two left-handed helical strands. The visualization of the substructure within the PHFs is rather variable and a model building approach has therefore been adopted, which has allowed the main features seen in the images to be interpreted. The subunit appears to have at least two domains in a radial direction and an axial extent of less than 5 nm. The whole structure can best be described as a twisted ribbon and indeed alkali treatment does untwist PHFs to give flat ribbons. The nature of the proposed model makes it most unlikely that the PHF is formed by a simple collapse of normal cytoskeletal elements, such as neurofilaments.     

Structure of mitochondrial F1-ATPase studied by electron microscopy and image processing

The structure of soluble F₁-ATPase (EC 3.6.1.3) has been investigated by computer analysis of individual molecular images extracted from electron micrographs of negatively stained particles. A total of 1241 images was interactively selected from several digitized micrographs and these images were subsequently aligned relative to different reference images. They were then submitted to a multivariate statistical classification procedure. We have focussed our attention on the main ‘hexagonal’ view which represents some 40% of our population of images. In this view, six masses are located on the outer region of the projection which are associated with the alpha and the beta subunits of the protein. A seventh mass is located close to the centre of the hexagon, but slightly off its exact midpoint. It has the shape of the letter V and its two legs point to two of the outer protein masses, or one alpha-beta subunit pair. The corner of the V has a density as high as those of the large subunits. Possible subunit arrangements and their consequences for the mechanism of ATP synthesis are discussed.     

Expression, purification and the 1.8 angstroms resolution crystal structure of human neuron specific enolase

Human neuron-specific enolase (NSE) or isozyme gamma has been expressed with a C-terminal His-tag in Escherichia coli. The enzyme has been purified, crystallized and its crystal structure determined. In the crystals the enzyme forms the asymmetric complex NSE x Mg2 x SO4/NSE x Mg x Cl, where "/" separates the dimer subunits. The subunit that contains the sulfate (or phosphate) ion and two magnesium ions is in the closed conformation observed in enolase complexes with the substrate or its analogues; the other subunit is in the open conformation observed in enolase subunits without bound substrate or analogues. This indicates negative cooperativity for ligand binding between subunits. Electrostatic charge differences between isozymes alpha and gamma, -19 at physiological pH, are concentrated in the regions of the molecular surface that are negatively charged in alpha, i.e. surface areas negatively charged in alpha are more negatively charged in gamma, while areas that are neutral or positively charged tend to be charge-conserved.     

Structural characterisation of neuronal voltage-sensitive K+ channels heterologously expressed in Pichia pastoris

Neuronal voltage-dependent K(+) channels of the delayed rectifier type consist of multiple Kv alpha subunit variants, which assemble as hetero- or homotetramers, together with four Kv beta auxiliary subunits. Direct structural information on these proteins has not been forthcoming due to the difficulty in isolating the native K(+) channels. We have overexpressed the subunit genes in the yeast Pichia pastoris. The Kv1.2 subunit expressed alone is shown to fold into a native conformation as determined by high-affinity binding of 125I-labelled alpha-dendrotoxin, while co-expressed Kv1.2 and Kv beta 2 subunits co-assembled to form native-like oligomers. Sites of post-translational modifications causing apparent heterogeneity on SDS-PAGE were identified by site-directed mutagenesis. Engineering to include affinity tags and scale-up of production by fermentation allowed routine purification of milligram quantities of homo- and heteroligomeric channels. Single-particle electron microscopy of the purified channels was used to generate a 3D volume to 2.1 nm resolution. Protein domains were assigned by fitting crystal structures of related bacterial proteins. Addition of exogenous lipid followed by detergent dialysis produced well-ordered 2D crystals that exhibited mostly p12(1) symmetry. Projection maps of negatively stained crystals show the constituent molecules to be 4-fold symmetric, as expected for the octameric K(+) channel complex.     

Two-dimensional crystalline sheets of Bacillus stearothermophilus 50S ribosomal particles.

Two-dimensional crystalline sheets of the large ribosomal subunit from Bacillus stearothermophilus have been obtained using a slightly modified procedure to that for growing three-dimensional crystals of the same material. The crystalline subunits are packed within monolayers in a relatively small unit cell, the dimensions of which are closely related to those observed for two forms of the three-dimensional crystals. The packing symmetry is p121, and the optical diffraction patterns of micrographs of negatively stained crystals extend to approximately 3.0 nm.