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At least 163 records · Page 9Linked to original sources
PubMed1997

Three-dimensional structure of the porcine gastric H,K-ATPase from negatively stained crystals.

A low-resolution three-dimensional model of membrane-bound H,K-ATPase from pig gastric mucosa has been reconstructed by electron microscopy and image processing of two-dimensional crystals in negative stain. The crystal formation is induced by magnesium and vanadate, which stabilize the E2 conformation of the enzyme. The unit cell, with a size of a = b = 123 A, gamma = 90 degrees, has tetragonal p4 symmetry. There are four separate alpha beta protomers within each unit cell. The high-contrast region is limited to the cytoplasmic part of the protein. The total volume of the observed asymmetric protein domain corresponds to a molecular mass of 80-90 kDa. It consists mainly of a large pear-shaped domain measuring 60 x 45 A2, with a height of 50 A as measured perpendicular to the membrane plane. A small stalk segment, 20 A in length, forms a connection to the transmembrane region.

Animals↗
PubMed2004

Conformational flexibility in recombinant measles virus nucleocapsids visualised by cryo-negative stain electron microscopy and real-space helical reconstruction.

Measles virus is a highly contagious virus that, despite the existence of an effective vaccine, is a major cause of illness and mortality worldwide. The virus has a negative-sense, single-stranded RNA genome that is encapsidated by the nucleocapsid protein (N) to form a helical ribonucleoprotein complex known as the nucleocapsid. This structure serves as the template for both transcription and replication. Paramyxovirus nucleocapsids are flexible structures, a trait that has hitherto hampered structural analysis even at low resolution. We have investigated the extent of this structural plasticity, using real-space methods to calculate three-dimensional reconstructions of recombinant nucleocapsids from cryo-negative stain transmission electron micrographs. Images of short sections of helix were sorted according to both pitch (the axial rise per turn) and twist (the number of subunits per turn). Our analysis indicates that there is extensive conformational flexibility within these structures, ranging in pitch from 50 Angstrom to 66 Angstrom, while twist varies from at least 13.04 to 13.44 with a greater number of helices comprising around 13.1 subunits per turn. We have also investigated the influence of the C terminus of N on helix conformation, analysing nucleocapsids after having removed this domain by trypsin digestion. We have found that this causes a marked change in both pitch and twist, such that the pitch becomes shorter, ranging from 46 Angstrom to 52 Angstrom, while more helices have a twist of approximately 13.3 subunits per turn. Our findings lead us to propose a mechanism whereby changes in conformation, influenced by interactions between viral or host proteins and the C terminus of N, might have a role in regulating the balance of transcription and replication during virus infection.

Measles virus↗
PubMed1988

Lateral segregation of sterol and channel proteins in the mitochondrial outer membrane induced by phospholipase A2: evidence from negative-stain electron microscopy using filipin.

The channel protein in the mitochondrial outer membrane of Neurospora crassa aggregates laterally into crystalline arrays by the action of phospholipase A2. When mitochondrial outer membranes are reacted with filipin and examined by negative-stain electron microscopy, filipin-sterol complexes are found everywhere on the membranes except on the crystalline channel arrays. This suggests that the channel-rich membrane domains may have a relatively low content of accessible sterol. It is proposed that in vitro segregation of protein and lipid membrane components by phospholipase A2 may reflect a mechanism by which the endogenous enzyme organizes the native mitochondrial membrane into functional domains.

Filipin↗
PubMed1985

Electron microscopy of influenza virus. A comparison of negatively stained and ice-embedded particles.

An electron microscopical study was made of the influenza virus, type B/Hong Kong, in the unstained, frozen, hydrated state after quench-freezing in cooled liquid ethane. The results are compared with data from negatively stained specimens. It is shown that cryo-electron microscopy confirms and extends the data obtained by conventional methods. In particular, the virus is shown to be circular in projection with no indication of icosahedral symmetry, the lipid membrane is clearly resolved as a bi-layer and it is demonstrated that the distribution of material within the bi-layer is non-uniform, with a shell of more electron dense material surrounding a less dense central region. Neuraminidase spikes are not clearly distinguished from haemaglutinin spikes. The diameter of the complete B/Hong Kong virus was estimated from cryo-micrographs as 1270(+/- 70) A. Some preliminary data for influenza virus type A/X31 are presented.

Freezing↗
PubMed1981

Molecular sieve in rat tubular basement membrane as revealed by negative staining.

Rat tubuli were isolated both by the method of Krisko and by a modified method of Cook and Pickering; renal tubular basement membrane (TBM) was isolated by sonic disruption and by the method of Carlson. Using electron microscopy after negative staining, TBM of rat kidney isolated under these different conditions was shown to be a fine meshwork. Strands of the meshwork were interwoven, apparently enclosing pores to make up whole basement membrane. These findings are compared with our previous observation that glomerular and alveolar basement membranes were made up of a similarly fine meshwork.

Albumins↗
PubMed1967

A study of newt mitotic chromosomes by negative staining.

A method is described for bursting single, selected mitotic cells on a fluid surface. Cells from cultures of newt heart tissue were burst on dilute solutions containing potassium and sodium with and without added calcium and also on dilute calcium chloride solution. The material was negatively stained with uranyl acetate or sometimes with ammonium molybdate or sodium phosphotungstate. The bodies of chromatids spread on NaCl/KCl solutions showed many parallel fibers about 150 A in diameter. Loops with a complex nodular structure were observed projecting from the sides and ends of chromatids. In calcium-containing solutions there was evidence of fiber coagulation; the chromatid body was more compact and laterally projecting fibers tended to be pulled out straight. Especially in the absence of calcium the chromosomal fibers had a nodular form and appeared to be composed of irregularly folded fibrillar elements. The question as to whether chromosomal fibers, which range in diameter from about 50 to 300 A, consist of single, folded threads or of two or more adjacent subunits is discussed.

Animals↗
PubMed1986

Structures attached to doublet microtubules of cilia: computer modeling of thin-section and negative-stain stereo images.

With a single set of positional coordinates for longitudinal and transverse attachment of the inner and outer rows of dynein arms with respect to the doublet microtubules of Tetrahymena ciliary axonemes, a computer model has been constructed at 4-nm resolution that reconciles negative-stain en face stereo images of arm and spoke positions to traditional images of tannic acid/glutaraldehyde-fixed sections. In this model, inner and outer arms correspond in substructure; both repeat with a 24-nm periodicity without stagger between rows, and a pair of arms is in exact alignment with the first spoke (S1) in each doublet spoke group. The model and the supporting micrographs suggest that each arm cycles in three dimensions and that, during cycling, the inner and outer arms move in opposite directions with respect to the center of subfiber A of the doublet (N). Attachment is off-center with respect to subfiber B of the adjacent doublet (N + 1), causing the sliding doublets to skew with respect to one another.

Adenosine Triphosphatases↗
PubMed1992

Structure-function relationships of the yeast fatty acid synthase: negative-stain, cryo-electron microscopy, and image analysis studies of the end views of the structure.

The yeast fatty acid synthase (M(r) = 2.5 x 10(6)) is organized in an alpha 6 beta 6 complex. In these studies, the synthase structure has been examined by negative-stain and cryo-electron microscopy. Side and end views of the structure indicate that the molecule, shaped similar to a prolate ellipsoid, has a high-density band of protein bisecting its major axis. Stained and frozen-hydrated average images of the end views show an excellent concordance and a hexagonal ring having three each alternating egg- and kidney-shaped features with low-protein-density protrusions extending outward from the egg-shaped features. Images also show that the barrel-like structure is not hollow but has a Y-shaped central core, which appears to make contact with the three egg-shaped features. Numerous side views of the structure give good evidence that the beta subunits have an archlike shape. We propose a model for the synthase that has point-group symmetry 32 and six equivalent sites of fatty acid synthesis. The protomeric unit is alpha 2 beta 2. The ends of each of the two archlike beta subunits interact with opposite sides of the two dichotomously arranged disclike alpha subunits. Three such protomeric units form the ring. We propose that the six fatty acid synthesizing centers are composed of two complementary half-alpha subunits and a beta subunit, an arrangement having all the partial activities of the multifunctional enzyme required for fatty acid synthesis.

Fatty Acid Synthases↗
PubMed1999

Ultrastructure of the eukaryotic aminoacyl-tRNA synthetase complex derived from two dimensional averaging and classification of negatively stained electron microscopic images.

Several aminoacyl-tRNA synthetases in higher eukaryotes are consistently isolated as a multi-enzyme complex for which little structural information is yet known. This study uses computational methods for analysis of electron microscopic images of the particle. A data set of almost 2000 negatively stained images was processed through reference-free alignment and multivariate statistical analysis. Interpretable structural information was evident in five eigenvectors. Hierarchical ascendant classification extracted clusters corresponding to distinct image orientations. The class averages are consistent with rotations around and orthogonal to a central particle axis and provide particle measurements: approximately 25 nm in height, 30 nm at the widest point and 23 nm thick. The results also provide objective evidence in support of the working structural model and demonstrate the feasibility of obtaining the three dimensional structure of the multisynthetase complex by single particle reconstruction methods.

Algorithms↗
PubMed1988

Further studies of HIV morphology by negative staining.

Thin-section studies of HIV-1- and HIV-2-infected cells were used to establish peak virus productivity and distribution of virus on and around infected cells. Maximum virus yields occurred 7 days after passage; cells at that stage were used as a source of virus for negative staining. Various methods of separating virus and cells were assessed: results showed that gentle homogenization in a Tenbroek-type homogenizer yielded considerably more virus than other techniques. Virus obtained in this way mainly appeared in the form of large clumps. Because of the large numbers of virus particles obtained it was possible to visualize what is probably the immature form of the virus. The inner component of this particle is spherical and, as is discussed, is a transient form proceeding to the now well established, mature, cone-shaped virus core.

Cell Line↗
PubMed1981

Attempted direct visualization of negatively stained amplified immune complex of synaptic acetylcholinesterase using cryoultramicrotomy sections.

An immunocytochemical method is proposed for the localization of synaptic acetylcholinesterase (AChE) on ultrathin frozen sections of the electric organ of the electric eel. The immune complex formed is amplified by a non-specific "sandwich" technique and visualized by negative staining. Definite white spots on synaptic cleft seem to correspond to basal lamina AChE molecules.

Acetylcholinesterase↗
PubMed1986

Three-dimensional reconstruction of negatively stained crystals of the Ca2+-ATPase from muscle sarcoplasmic reticulum.

The structure of the Ca2+ transport ATPase from rabbit skeletal muscle sarcoplasmic reticulum has been determined to 25 A resolution by three-dimensional image reconstruction of crystalline membrane tubules induced through exposure to Na3VO4 and preserved for electron microscopy in negative stain. The crystalline arrays have projection symmetry p2 and consist of chains of Ca2+-ATPase dimers arranged in a right-handed helix. The density map shows protein features that project from the membrane surface into the cytoplasm. The luminal side of the membrane tubules is featureless, presumably because very little of the Ca2+-ATPase molecule projects into the luminal space. The cytoplasmic region of the Ca2+-ATPase molecule is pear-shaped, with a lobe oriented nearly parallel to the axis of the dimer ribbons, about 16 A above the surface of the membrane bilayer. The structure seen in the maps has a volume of 71,000 A3, corresponding to a molecular weight of 57,000. The two Ca2+-ATPase profiles that constitute a dimer are connected by a stain-excluding bridge that is oriented parallel with the axis of the tubule at a height of about 42 A above the surface of the bilayer.

Animals↗
PubMed1990

The structure of microtubule ends during the elongation and shortening phases of dynamic instability examined by negative-stain electron microscopy.

Microtubules (MTs) are dynamic polymers that can exist in phases of elongation and rapid-shortening at steady-state. These phases have been observed in vitro and in living cells, and this property of MTs has been termed 'dynamic instability'. The purpose of this study was to use negative-stain electron microscopy (EM) to test if there are structural differences between the ends of MTs in the elongation and shortening phases, which could provide insight into the mechanisms of dynamic instability. MTs in the elongation phase were obtained by seeding either highly purified porcine brain tubulin (PC-tubulin) or tubulin containing microtubule-associated proteins (MTP), from isolated Tetrahymena axonemes. The results are that, in addition to intact cylindrical MTs, a significant fraction of the tubulin polymer in the elongation phase occurred as sheets of parallel protofilaments, as found in previous investigations with self-assembled MTs. Therefore, sheet formation is an intrinsic property of MT assembly that does not depend on the tubulin purity or the method of nucleation. Also, since sheets lack helical symmetry, at least a fraction of tubulin polymers seeded from axonemes did not assemble by helical addition of tubulin dimers to the ends, an assumption often made in mathematical models of dynamic instability. Sheets and intact MTs that were seeded from isolated axonemes, emanated both from the intact MT wall of the axoneme A-subfiber and from the incomplete wall of the B-subfiber. Therefore, axoneme seeds do not provide a homogeneous nucleation site for tubulin growth, or produce a homogeneous population of tubulin polymers under our conditions. Previous evidence has indicated that MT disassembly can occur by a segmental release of tubulin oligomers from the ends and at sites along the length of MTs. However, these studies were performed with MTP, and disassembly was induced by cold depolymerization. We examined MT shortening under conditions that closely represent shortening via dynamic instability, namely isothermal dilution at 37 degrees C of self-assembled MTs. This was compared with the morphology of cold-disassembled MTs. The cold-depolymerization of MTs composed of MTP showed rings and protofilament curls as previously observed using similar methods. Surprisingly, cold-depolymerization of MTs assembled from PC-tubulin induced not only shortening, but also the opening of a large fraction of MTs into sheets, suggesting that the MT lattice contains a cold-labile seam. Under conditions that mimic stochastic shortening, MTs were intact, closed cylinders with ends that were approximately blunt. Therefore, rapid shortening occurs at the ends of the MT, without a long-range disruption of the MT wall. In conclusion, MTs in the elongation phase can have highly irregular ends and need not elongate by a helical assembly process. Conversely, MTs in the shortening phase can have relatively blunt, even ends and can depolymerize in a relatively uniform fashion.

Animals↗
PubMed1977

Infantile Gaucher's disease: neuropathology, acid hydrolase activities and negative staining observations.

A case of infantile Gaucher's disease with widespread cerebral degenerative changes is presented. Neuropathological features included perivascular Gaucher cell infiltrates accompanied by periadventitial fibrogliosis, focal neuronophagia, Purkinje cell vacuolation and diffuse astrocytosis of cerebellar white matter, brain stem and spinal paracentral grey matter. Gaucher cell tubules were isolated from formalin fixed material. Negative staining confirmed the appearance of a coiled helical structure, 200--400 A in diameter composed of microfibrils. A relative deficiency of acid beta-glucosidase was found in spleen homogenate accompanied by a considerable increase in acid phosphohydrolase, N-acetylglucosaminidase, beta-glucuronidase and beta-galactosidase.

Brain↗
PubMedDate not supplied

Electron microscopy of tRNA crystals. I. Thin crystals negatively stained with uranyl acetate.

The first attempt to study crystal structures of tRNA by electron microscopy is described. Sufficiently thin crystals were prepared from yeast tRNAphe. The thickness of the thinnest was estimated at 130 A corresponding to a bilayer of the molecules. The L-shaped structure seemed to be maintained even after the negative staining with uranyl acetate. Optically filtered images from electron micrographs were compared with those simulated from the drawing of the molecular model by optical transform. The results suggest that the observed images reflect the real molecular arrangements within the crystal lattice although the shape of tRNA molecules seems to be somewhat modified by the uneven staining.

Crystallography↗
PubMed1985

Improved negative staining of microfilament arrangements in detergent-extracted Physarum amoeboflagellates.

A motile, lamellipodium-like structure, the ridge, forms as amoeboflagellate cells of Physarum polycephalum release from a substratum and begin swimming in fluid. Actin microfilaments form a distinct laminar core within the ridge; they are seen as a sparse, disordered meshwork in cytoskeletons prepared by conventional methods using uranyl acetate negative staining [10]. Preservation and visualization of these filaments and their arrangements improved considerably when cytoskeletons were imaged with phosphotungstic acid buffered with ammonium hydroxide (PTA(NH4]. Microfilaments within ridge cytoskeletons were found to form loose bundles and criss-crossing, 'meshwork' arrays several layers deep. Differences could be detected in morphology and detailed arrangement of microfilaments within cytoskeletons prepared in the presence of phalloidin. PTA(NH4) may be useful for studies of cytoskeletal elements and their rearrangements in dynamic, motile regions of cells.

Cell Cycle↗
PubMed2003

Three-dimensional structure of the Neisseria meningitidis secretin PilQ determined from negative-stain transmission electron microscopy.

The PilQ secretin from the pathogenic bacterium Neisseria meningitidis is an integral outer membrane protein complex which plays a crucial role in the biogenesis of type IV pili. We present here the first three-dimensional structure of this type of secretin at 2.5-nm resolution, obtained by single-particle averaging methods applied to the purified protein complex visualized in a negative stain. In projection, the PilQ complex is circular, with a donut-like appearance. When viewed from the side it has a rounded, conical profile. The complex was demonstrated to have 12-fold rotational symmetry, and this property was used to improve the quality of the density map by symmetry averaging. The dominant feature of the structure is a cavity, 10 nm deep, within the center of the molecule. The cavity is funnel-shaped in cross section, measures 6.5 nm in diameter at the top of the complex, and tapers to a closed point, effectively blocking formation of a continuous pore through the PilQ complex. These results suggest that the complex would have to undergo a conformational change in order to accommodate an assembled pilus fiber of diameter 6.5 nm running through the outer membrane.

Biological Transport↗