Ordered membrane particles in rhabdomeric microvilli of the housefly (Musca domestica L.)

Rhabdomeric microvilli of the housefly were freeze-fractured (FF) and thin sectioned (TS) for ultrastructural examination. Ordered files of closely packed membrane particles (82 Å wide, 250 Å long) were seen (FF) on the microvillar membrane (usually E face). The long axis of each particle was canted about 45° to that of the microvillus. Occasionally particles in this array appeared on the P face. It is hypothesized that ordered particles may represent either a photopigment precursor stock, a second photolabile pigment, or the newly discovered sensitizing, UV-absorbing, photostable visual pigment. In the underlying membrane leaflet (P face) were found spherical (85 Å diameter) unoriented particles in a concentration of about 6,000/μm². The size, shape and density of these structures are compatible with those of rhodopsin particles. These particles also covered the basal area of each microvillus. The findings from TS material were difficult to correlate with those from FF replicas. At high magnification the former showed that the plasma membrane of the transected microvillus is composed of spherical, hollow subunits (averaging 43 Å diameter), sometimes fused to form double, 86 Å units. These substructures were closely packed and continuous around the microvillus. This beaded plasma membrane, in rare cases, was doubled around the microvillus. In other instances the plasma membranes were continuous between neighboring microvilli. The physiological implications of these ultrastructural features are discussed.     

Three-dimensional reconstruction of the flagellar hook from Caulobacter crescentus

The structure of the bacterial flagellar hook produced by a mutant of Caulobacter crescentus was studied by electron microscopy, optical diffraction, and digital image processing techniques. The helical surface lattice of the hook is defined by a single, right-handed genetic helix having a pitch of about 23 Å, an axial rise per subunit of 4 Å and an azimuthal angle between subunits of 64·5 °. The lattice is also characterized by intersecting families of 5-start, 6-start and long-pitch 11-start helices. These helical parameters are remarkably similar to those determined for the flagellar filaments from several strains of gram-negative bacteria. The technique of three-dimensional image reconstruction (DeRosier & Klug, 1968) was applied to nine of the better preserved specimens and the diffraction data from five of these were correlated and averaged and used to generate an average three-dimensional model of the hook. The pattern of density modulations in the three-dimensional model is suggestive of an elongated, curved shape for the hook subunit (100 Å × 25 Å × 25 Å). The subunits are situated in the lattice of the polyhook such that their long axes are tilted about 45 ° with respect to the hook axis. The subunits appear to make contact with each other along the 6-start helices at a radius of 80 Å and also along the 11-start helices at a radius of 65 Å. Few structural features are revealed at radii between 15 å and 45 Å and, therefore, we are unable to decide to what extent the hook subunits extend into this region. The most striking characteristic of the model is the presence of deep, broad, continuous 6-start helical grooves extending from an inner radius of about 50 Å to the perimeter of the particle at 105 Å radius. Normal hooks usually appear curved in electron micrographs and sometimes so are the mutant hooks; the prominent 6-start grooves appear to allow for bending with minimal distortion of matter in the outer regions of the hook. A round stain-filled channel about 25 Å in diameter runs down the center of the polyhook. Such a channel supports a model for flagellar assembly in which flagellin subunits travel through the interior of the flagellum to the growing distal end of the filament.     

The double-helical molecular structure of crystalline b-amylose

The crystal structure of the B-polymorph of amylose appears to be based on double-stranded helices. The individual strands are in a right-handed six-fold helical conformation repeating in 20.8 Å and are wound parallel around each other. The steric disposition of O-6 is gt. The double helices pack in a hexagonal unit-cell (a  b  18.50 Å, c (fiber repeat)  10.40 Å, γ  120°), with two helices (12 d-glucose residues) per cell. The helices are packed antiparallel and leave an open channel within a hexagonal array that is filled with water molecules. The reliability of the structure analysis is indicated by R  0.22. The structure of B-amylose is consistent with the diffraction diagrams of B-starches and accounts for the physical properties of such starches.     

X-ray diffraction and electron microscope studies on the structure of bacterial F pili.

F pili are hollow cylinders with 80 Å outer diameter and 20 Å inner diameter. Both X-ray fibre diffraction and optical diffraction of electron micrographs show a strong layer-line corresponding to a spacing of 32 Å, to which a J₄ Bessel function is assigned on the basis of the optical diffraction. X-ray diffraction patterns show near-meridional intensity on a layer-line corresponding to a spacing of 12.8 Å, to which a J₁ Bessel function is assigned. Mass per length measurements on unstained specimens in the scanning transmission electron microscope give 3000 daltons/Å, indicating that the 11,200 dalton pilin subunits are 3.7 Å apart along the axial direction of the pili. These observations show that the pilus structure can be represented as four coaxial helices of pitch 128 Å with the pilin subunits elongated and overlapping along the line of these helices. Each of these helices of subunits is translated axially with respect to its neighbour, to give a basic helix of 3.6 units per turn of 12.8 Å pitch. Radial electron density calculations indicate a 50 Å diameter girdle of hydrophobic amino acids between the inner and outer diameters of the protein shell. A molecular model of the structure at low resolution is presented.     

Bacteriophages of Clostridium saccharoperbutylacetonicum

The fine structure of phage HM 2 (group I) active on Clostridium saccharoperbutylacetonicum was studied by an electron microscopy with a negative-staining technique, and compared with those of more conventional types, phages HM 3 (group II) and HM 7 (group III), whose tails were clearly observed by a shadow-casting technique. This study revealed that phage HM 2 had an intricate tail which was not observed by a shadow-casting technique.

Phage HM 2 has an icosahedral head about 450 Å in diameter and a non-contractile tail about 300 Å long. The distal 130 Å of the tail axis has a width of 80 Å which is wider than the upper portion of the tail (50 to 60 Å). The distal enlargement is not seen in the hollow tail. Twelve fibrous-shaped appendages are attached symmetrically at the upper portion of tail axis and extend toward the distal base of the tail. Their length is a little shorter than 300 Å. They combine with divalent cations in the phage dilution medium, and also adsorb the host cell debris.

Phage HM 3 has an icosahedral head about 770 Å in diameter and a tail about 1000 Å long and 150 Å wide with contractile sheath. Phage HM 7 has an icosahedral head about 750 Å in diameter and a long non-contractile tail about 2000 Å long and about 120 Å wide with forked tip.

The structure of the tail of phage HM 2 is quite different from those of phages HM 3 and HM 7 hitherto described and those of the various phages of other bacteria.     

The three-dimensional structure of mitochondrial aspartate aminotransferase at 4.5 A resolution

An X-ray crystallographic study at 4.5 Å resolution has been carried out with triclinic crystals of chicken mitochondrial aspartate aminotransferase.In the electron density map, the enzyme is clearly visible as an isologous α₂-dimer (105 Å × 60 Å × 50 Å) in which the subunits are associated about a molecular 2-fold axis. Each subunit of dimensions 70 Å × 50 Å × 40 Å contains at least seven helices, one of which is about 50 Å long.Difference maps have revealed the positions of the pyridoxyl and the phosphate moieties of the coenzyme as well as the general substrate binding area. The active sites are on opposite sides of the dimer, about 30 Å apart and close to the intersubunit boundary, so that probably both subunits contribute to each active site. An isolated chain segment, passing in front of the active site and ending in contact with the neighbouring subunit is interpreted as one of the chain termini.     

FINE STRUCTURE OF CHROMOSOMES

Electron micrographs of staminate hair cells of Tradescantia reflexa indicate that early prophase chromosomes are composed of a number of helically arranged chromonemata. Favorable preparations reveal as many as 64 identifiable subsidiary strands, assumedly arranged as intertwined pairs to form a hierarchy of pairs of pairs. The helices of the smallest discernible units have a diameter of about 125 A, with highly electron-scattering material disposed peripherally around a less dense "core." The wall of this peripheral ring has a thickness of about 40 A, and apparently represents another pair of coiled threads surrounding a 40 A central axis. The implications of the findings are discussed briefly.     

Ultrastructural organization of heterochromatin within sea urchin sperm nuclei

Chromatin within swollen or lysed isolated sperm nuclei of the sea urchin, Strongylocentrotus purpuratus, was examined by electron microscopy. Spread preparations of lysed sperm nuclei demonstrated dense aggregates of nondispersed material and beaded filaments radiating from these aggregates. These beaded fibers are similar in size and appearance to the “beads-on-a-string” seen as characteristic of chromatin spreads from numerous interphase nuclei. The beads are nucleosomes that have an average diameter of 130 Å. The interconnecting string is 40 Å indiameter and corresponds to the spacer DNA. In thin sections of swollen nuclei the sperm chromatin appears to be composed of 400 Å superbeads that are closely apposed to form 400 Å fibers. As the chromatin disperses, the superbeads are seen to be attached to one another by chromatin fibers of 110 Å diameter. In thin sections, the 400 Å superbeads appear to disperse directly into the 110 Å fibers with no intervening structures. This work demonstrates that the heterochromatin in Strongylocentrotus purpuratus sperm nuclei is composed of nucleosomes that form 100 Å filaments that are compacted into 400 Å superbeads. The superbeads coalesce to give the morphological appearance of 400 Å fibers.     

The periodicity and architecture of lipid retained and extracted lung surfactant and its origin from multilamellar bodies.

Lipid and carbohydrate retaining procedures were utilized to retain these components of surfactant in thin section. The origin of tubular myelin surfactant from multilamellar bodies was postulated on morphological evidence to be : an expansion of lamellae in pairs caused by the absorption of a homogeneous, probably carbohydratebased, material of undetermined origin; immediately after the pairs expanded to 438 Å, the lamella (lipid bilayer) that was most peripheral in the multilamellar body underwent a molecular rearrangement to form the two cross lipid bilayers; the four lipid bilayers formed a square tubule 413 Å of side and 0.16–1.8 μ long. The homogeneous material included within each tubule (the surfactant matrix) did not completely fill the intratubular space, but a centrally located, spherical, 57 Å diameter, electron transparent, area was observed. The 413 Å square tubules were separated on all sides by a 25 Å fatty acid tail area. The phospholipid head areas were 46 Å and the surfactant matrix was 132 Å between the electron transparent, center area and the phospholipid heads layer.Extraction with several organic solvents was also studied: slight ethanol extraction—alteration of surfactant matrix and loss of transparent, central area. Moderate ethanol extraction—alteration of surfactant matrix to form artifactual tubules and filaments, disruption of lipid bilayers. Severe extraction—ethanol produced loss of integrity of both matrix and lipid bilayers; vinylcyclohexane dioxide or methacrylate produced complete loss of all lipid bilayers, but surfactant matrix retained tubular configuration ; DMP-30 produced complete loss of lipid bilayers, and although most of the matrix material was retained, all areas appeared fractured and randomly oriented.     

Folding of Globular Proteins by Energy Minimization and Monte Carlo Simulations with Hydrophobic Surface Area Potentials

We describe an efficient method to calculate analytically the solvent accessible surface areas and their gradients in proteins for empirical force field calculations on serial and parallel computers. In an application to the small three helix bundle protein Er-10, energy minimizations and Monte Carlo simulations were performed with the empirical ECEPP/2 force field, which was extended by a protein solvent interaction term. We show that the NMR structure is stable when refined with the force field including the protein solvent interaction term, but large structural deviations are observed in energy minimization in vacuo. When we started from random structures with preformed helices and maintained the helical segments by dihedral angle constraints, the final structures with the lowest energies resembled the native form. The root-mean-square deviations for the backbone atoms of the three helices compared to the experimentally determined structure was 3 Å to 4 Å.     

Development of Haemogregarina boueti in the toad Bufo regularis*

SYNOPSIS. Haemogregarina boueti França, 1910, was found to be the commonest blood parasite in the common toad, Bufo regularis Reuss, in Egypt. The rate of infection was about 30% (of 689 toads examined). In properly fixed blood films, the parasites were almost exclusively intraerythrocytic. Most characteristic was the encapsulated “elongate” form averaging 22.3 by 6 μ with a more-or-less central nucleus and a pointed, slightly bent, posterior end. Infected red cells were conspicuously hypertrophied and their nuclei were markedly displaced and frequently broken into 2-4 parts. Young and growing blood forms as well as two types of hepatic schizonts are described for the first time. Schizonts of the first type develop in hepatic cells, are 28–30 μ in diameter and produce numerous elongate oval merozoites about 8 × 2.2 μ radially arranged around a residual body about 10 μ in diameter. Schizonts of the second type start their growth in erythrocytes but later complete their development as free bodies in the liver sinusoids. When mature, they are 32–35 μ in diameter and produce a larger number of thin merozoites about 8 × 1.5 μ, surrounding a larger residual body about 19 μ in diameter.     

MORPHOLOGY OF THE OMMATIDIA OF THE COMPOUND EYE OF LIMULUS

The sensory portion of the ommatidium of the compound eye of Limulus has been studied with the electron microscope. In axial longitudinal section the rhabdom appears to be made up of small polygons, and in transverse section the rhabdom appears as a banded structure of dark lines. Thus in three dimensions the rhabdom resembles a honeycomb composed of tubular units, the long axes of which lie in transverse planes and are oriented perpendicular to the retinula cell's contours. The tubular units, which are about 140 mµ in diameter in Limulus (70 mµ in diameter in the spider and Scutigera), are microvilli of the borders of the retinula cells. The walls of these microvilli are continuous with fine linear structures (membranes) in the cytoplasm of the retinula cells. In transverse sections of the ommatidium oval bodies interpreted as mitochondria are observed in an annular zone at the tips of the rhabdom's rays. These mitochondria, which are 2 to 10 µ in diameter, are crowded with irregular closed outlines about 100 mµ in diameter. Possible functions of components of the ommatidium are discussed.     

Spatial organization of catalase proteins

The three-dimensional structure of the heme-containing fungal catalase fromPenicillium vitale (m.m. 2,80,000) has been studied by X-ray analysis at 2.0 A resolution. The molecule is tetramer, each subunit contains 670 aminoacid residues identified to construct “X-ray” primary structure. The subunit is built of three compact domains and their connections. The first domain of about 350 residues contains aβ-barrel flanked by helices, the second domain of 70 residues is formed by four helices and the third one is composed of 150 residues and is topologically similar to flavodoxin. The active site including heme is deeply buried near theβ-barrel. A comparison of the structure of catalase fromPenicillium vitale with that of beef liver catalase revealed very close structural homology of the first and the second domain, but the third domain is entirely absent in beef liver catalase. A catalase from thermophillic bacteriaThermus thermophilus (m.m. 2,10,000) has been first isolated, crystallized and studied by X-ray analysis. Crystals are cubic, space group is P2₁3, a = 133.4 Å. The molecule is a hexamer with trigonal symmetry 32. The electron density map at 3 Å resolution made it possible to trace the polypeptide chain. The main structural motif is formed by four near parallel helices. There is no heme inThermus thermophilus catalase, the active site is between the four helices and contains two manganese ions.     

Elektronenmikroskopische Untersuchung alternder Zellen von Pseudomonas rhodos

During a 10 day-incubation on agar surfaces at 30°C, cells of the gram-negative soil bacterium Pseudomonas rhodos pass through three phases distinguishable by physiological and morphological criteria. When viewed by electron microscopy, typically “rolled” mesosomes could frequently be observed in young cells. In aged cells instead, loosely rolled or stretched-out, flattened tubules could be discerned, presumed to be degenerate mesosomes. Tubular flattened structures have been isolated from these cells by lysozyme treatment or sonication and were concentrated by differential centrifugation. Electron micrographs of these preparations showed long, straight tubules which sometimes appeared sealed at one end. Their width was 34±5 nm. They contained a lining of material, which could be digested by trypsin leaving behind an electron-transparent matrix. In rare cases, isolated tubules showed a periodic fine structure composed of ellipsoidal subunits. Optical diffraction analysis yielded a lattice consisting of subunits arranged in helices of pitch-angle 27°; the unit cell dimensions were shown to be 112×56 Å. Owing to their sensitivity to trypsin, components of the regular lattice are supposed to consist of protein. It is postulated that these protein components are layered onto a tubular membrane. These tubules are clearly distinguishable by their shape and fine structure from the periodic structure of a P. rhodos cell wall layer, which exhibits a tetragonal pattern, and also from polyheads and polysheaths of defective bacteriophages. Their possible origin from intact mesosomes is discussed.     

The Life Cycle of Ganymedes oaklandi n. sp., an Acephaline Gregarine of Gammarus fasciatus (Say)*

SYNOPSIS. Ganymedes oaklandi n. sp. (Ganymedidae) from the intestine of Gammarus fasciatus (Say) is described, and its life cycle is given. In the trophic stage the gregarine is acephaline, with an elongated cylindrical body slightly tapered anteriorly, and is up to 298 × 64 μ. The gametocysts are up to 121 × 99 μ, and are covered with a secreted thick sticky gelatinous coat. The spores are released from the gametocyst by rupturing of the cyst wall. The spore body is spherical, approximately 6 μ in diameter, and has 4 episporal rays about 20 μ long.     

Structure of the cytochrome c oxidase dimer electron microscopy of two-dimensional crystals

We have studied the structure of beef heart mitochondrial cytochrome c oxidase dimers by image-processing of electron micrographs of the vesicle crystal form. Specimens were prepared by different procedures, which contrast different features of the crystals. Heavy-atom shadowing of freeze-dried crystals contrasts the exterior or M-side surface (mitochondrial matrix-side) and reveals a 100 Å long ellipsoidal dimer oriented with its long axis in the (?1, 1) direction of the 95 Å × 125 Å rectangular unit cell. The M-side surface structure correlates well with the intra-bilayer structure revealed by contrast matching extra-bilayer protein with glucose. Frozen suspensions of vesicle crystals fracture predominantly along hydrophilic surfaces revealing the interior C-side (mitochondrial cytoplasm-facing surface) of vesicle crystals. The C-side surface revealed in shadowed replicas of fracture surfaces shows the ends of the dimers furthest from the bilayer surface; they consist of two structural domains separated by 70 to 80 Å. We present a new interpretation of the structure of the cytochrome oxidase dimer based on these data and on the y-shaped monomer structure described by Fuller et al. (1979). A cytochrome oxidase dimer is formed from two y-shaped monomers joined along one set of identical M-domain arms with the other arms approximately 70 Å apart along a unit cell diagonal in the (?1, 1) direction. The arms of the monomers lie within and perpendicular to the phospholipid bilayer, and they protrude approximately 25 Å beyond the bilayer surface on the M-side. The y tails represent the C-side domains, which are closely apposed across the dimer 2-fold axis near the C-side bilayer surface. Further away from the bilayer surface, C-side domains split away from one another forming a large cleft.     

Scanning electron microscopy of sporulating cultures of the myxomycete Physarum polycephalum

Scanning electron microscopy of sporulating cultures of Physarum polycephalum shows in detail the fine structure of the stalk, peridium, capillitia and spores. The peridium has a wrinkled structure and funnel-like cavities (5-10 μ in diameter) which are the capillitia openings. The capillitia are a network of tubes with differing diameters. The capillitia are covered with fine granules, and contain various amounts of bead-like granules of up to 2 μ in diameter. The spherical spores (10 μ in diameter) are covered with fine granules similar to those covering the capillitia.     

Structure of glycolate oxidase from spinach at a resolution of 5.5 Å

The crystal structure of glycolate oxidase from spinach has been determined to 5.5 Å resolution, using two isomorphous heavy-atom derivatives and their anomalous contributions. In the electron density map the boundaries of the octameric molecules are clearly seen. The subunit molecular weight is 37,000. Two protomers are in very close contact around one of the crystallographic 2-fold axes. Four such dimers are in contact around the 4-fold axis, so that the glycolate oxidase molecules are arranged as octamers with 422 symmetry in the crystal lattice. The roughly spherical octameric molecules have a diameter of approximately 100 Å. These octamers are arranged in a network, such that large solvent channels, approximately 60Å in diameter, pass right through the crystal lattice.The secondary structure of two-thirds of the subunit density has been interpreted in terms of eight consecutive β strand-α-helix units forming a cylinder very similar to the structure of triose phosphate isomerase. This interpretation is based on the very characteristic arrangement of the eight helices which form such a cylinder. The binding site of a substrate analogue, thioglycolate, has been localized in a deep cleft of the subunit at one end of the βα-barrel close to its axis.     

RNA sequences specifically associated with mouse intracisternal A particles.

Electron microscopic examination of the histone H1-depleted, folded genomes of Drosophila melanogaster reveals that they are composed of long cylindrical cables of about 100 Å diameter. Limited single-strand nicking with DNAase I relaxes the 100 Å fibers to a “beads-on-a-string” structure, showing the nucleosomes and internucleosome DNA.Based on these results and other available data, we have constructed a detailed space-filling model for the higher order DNA coiling in chromatin, starting with the symmetrical nucleosome core previously described (Weintraub, Worcel and Alberts, 1976). The model defines the path of the DNA helix and the nucleosome arrangement along the DNA coil for both the 100 Å and the 200–300 Å fibers.Following Sobell et al. (1976), we believe that the DNA is coiled in the 100 Å nucleofilament in a uniform left-handed supercoil of about 90 base pairs (bp) per turn and 47 Å pitch; the 140 bp symmetrical nucleosome cores align themselves along this uniform DNA superhelix so that the isologous outer surfaces of adjacent nucleosomes touch and the internucleosome spacer DNA coils between them. A few single-strand discontinuities [about one nick per 85 kilobases (kb); Benyajati and Worcel, 1976] in the H1-depleted 100 Å fiber can thus relax the negatively supercoiled internucleosome DNA generating the “beads-on -a-string” appearance.We propose that histone H1 binds to the 100 Å diameter superhelix and coils it into tightly packed, 110 Å pitch super-superhelices (“solenoids;” Finch and Klug, 1976) of variable diameter (between 200–300 Å). In our model, the “thick” 200–300 Å fiber is stabilized at metaphase by histone H1-H1 heterologous interactions between adjacent helical turns of the nucleofilament, and the internucleosome spacer DNA is located on the outside. Symmetry considerations demand that changes in the length of the repeat should lead to variations in the number of nucleosomes per helical turn and in the handedness of these turns in the 200–300 Å metaphase fiber.     

Retinal-tectal connections in the embryonic chick: evidence for regionally specific cell surface components which mimic the pattern of innervation.

The chorion surface in the eggs of the annual fishes Cynolebias melanotaenia and C. ladigesi contains an elaborate, three-dimensional species-specific pattern. Two concentric layers form the chorion. The pattern resides in the outer layer, the secondary envelope. It consists of closely packed tubules about 250 Å in diameter. A coat of electron dense “fuzzy” material increases this to 475 Å. The inner layer, the primary envelope, of uniformly low electron density possesses no obvious substructure. Oogenesis is divided into six stages. The oocyte increases in size from 10–20 μm in Stage 1 to 250 μm in Stage 3, 600 μm in Stage 4, and attains maximal size of 900 μm by Stage 6. Massive inclusions of protein and lipid yolk accumulate during Stages 4 and 5. Zone 1, one of the three zones of the primary envelope, first appears late in Stage 2. During Stage 3, Zone 1 is completed and Zone 2 appears between the oocyte surface and Zone 1. The oocyte cytoplasm increases in complexity. Material similar to Zone 1 (light, fibrillar) and Zone 2 (dark, compact) is present in the RER, Golgi, derivative vesicles, and apical pits. Micropyle formation also commences. The oocyte secretes Zone 3 during Stage 4 as thin filaments which consolidate into a highly ordered, transitional structure composed of tangentially oriented bundles of interwoven filaments. These partially fuse during Stage 5 except for fenestrations through which oocyte and follicle cell microvilli pass. Complete fusion during Stage 6 produces a continuous layer. Follicle cells retain an unspecialized structure from Stages 1 through 4. Secondary envelope material accumulates in the RER of the follicle cells during Stage 5. It is secreted and deposited during Stage 6.