PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “Cryoelectron Microscopy”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 37 records · Page 2Linked to original sources
PubMed1999

Capsid structure of simian cytomegalovirus from cryoelectron microscopy: evidence for tegument attachment sites.

We have used cryoelectron microscopy and image reconstruction to study B-capsids recovered from both the nuclear and the cytoplasmic fractions of cells infected with simian cytomegalovirus (SCMV). SCMV, a representative betaherpesvirus, could thus be compared with the previously described B-capsids of the alphaherpesviruses, herpes simplex virus type 1 (HSV-1) and equine herpesvirus 1 (EHV-1), and of channel catfish virus, an evolutionarily remote herpesvirus. Nuclear B-capsid architecture is generally conserved with SCMV, but it is 4% larger in inner radius than HSV-1, implying that its approximately 30% larger genome should be packed more tightly. Isolated SCMV B-capsids retain a relatively well preserved inner shell (or "small core") of scaffolding-assembly protein, whose radial-density profile indicates that this protein is approximately 16-nm long and consists of two domains connected by a low-density linker. As with HSV-1, the hexons but not the pentons of the major capsid protein (151 kDa) bind the smallest capsid protein (approximately 8 kDa). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed cytoplasmic B-capsid preparations to contain proteins similar in molecular weight to the basic phosphoprotein (approximately 119 kDa) and the matrix proteins (65 to 70 kDa). Micrographs revealed that these particles had variable amounts of surface-adherent material not present on nuclear B-capsids that we take to be tegument proteins. Cytoplasmic B-capsids were classified accordingly as lightly, moderately, or heavily tegumented. By comparing the three corresponding density maps with each other and with the nuclear B-capsid, two interactions were identified between putative tegument proteins and the capsid surface. One is between the major capsid protein and a protein estimated by electron microscopy to be 50 to 60 kDa; the other involves an elongated molecule estimated to be 100 to 120 kDa that is anchored on the triplexes, most likely on its dimer subunits. Candidates for the proteins bound at these sites are discussed. This first visualization of such linkages makes a step towards understanding the organization and functional rationale of the herpesvirus tegument.

Capsid↗
PubMed2005

Cryoelectron microscopy of vitrified sections: a new challenge for the analysis of functional nuclear architecture.

Cryoelectron microscopy of vitrified sections has become a powerful tool for investigating the fine structural features of cellular compartments. In the present study, this approach has been applied in order to explore the ultrastructural morphology of the interphase nucleus in different mammalian cultured cells. Rat hepatoma, Chinese hamster ovary and Potorus kidney cells were cryofixed by high-pressure freezing and the cryosections were examined at low temperature by transmission electron microscopy. Our results show that while the contrast of nuclear structural domains is remarkably homogeneous in hydrated sections, some of them can be recognised due to their characteristic texture. Thus, condensed chromatin appears finely granular and the perichromatin region contains rather abundant fibro-granular elements suggesting the presence of dispersed chromatin fibres and of perichromatin fibrils and granules. The interchromatin space looks homogeneous and interchromatin granules have not been identified under these preparative conditions. In the nucleolus, the most striking feature is the granular component, while the other parts of the nucleolar body, which appear less contrasted, are difficult to resolve. The nuclear envelope is easily recognisable with its regular perinuclear space and nuclear pore complexes. Our observations are discussed in the context of results obtained by other, more conventional electron microscopic methods.

Animals↗
PubMed1996

Three-dimensional reconstruction of Macrobdella decora (leech) hemoglobin by cryoelectron microscopy.

Macrobdella decora hemoglobin was observed in vitreous ice by cryoelectron microscopy and subjected to three-dimensional reconstruction by the method of random conical tilt series. The refined volume has a resolution of 40 A and a D6 point-group symmetry. Its architecture, with its hexagonal bilayer appearance, resembles those of Lumbricus terrestris (oligochaete) and Eudistylia vancouverii (polychaete). When the reconstruction volume is viewed along its sixfold axis, the vertices of the upper hexagonal layer are rotated 16 degrees clockwise compared to those of the lower layer. In agreement with the "bracelet" model of Vinogradov et al., a central linker complex is decorated by 12 hollow globular substructures. The linker complex is made up of a central hexagonal toroid linked by 12 c5 connections to two bracelets of c3 connections, which are themselves linked via six c4 connections. The portion of the hollow globular substructure corresponding to the dodecamer of globin chains has a local pseudo threefold symmetry and is composed of three elongated structures visible when the volume is displayed at high threshold. The main difference between Macrobdella, Lumbricus, and Eudistylia hemoglobins is the presence in Macrobdella of a central hexagonal toroid instead of a compact flat hexagonal structure.

Animals↗
PubMed1994

Cryoelectron microscopy of macromolecular complexes.

Although there are many macromolecular complexes which play extremely important roles in biology, and despite continued progress in X-ray crystallographic and NMR methods, it is still very difficult to obtain atomic level structural information about such large assemblies. It is now clear that a powerful approach is to combine structural information obtained at different levels. Cryoelectron microscopy of frozen-hydrated samples together with computer based 3-D reconstruction can give structural information at the quaternary level. This can then be combined with atomic level structures of individual components, obtained by X-ray crystallography or NMR to build-up a detailed picture of the overall architecture of the complexes and of the interactions between the components. In our laboratory we are particularly interested in developing the complementarity between the different structural approaches. The aim of this short review is to briefly present our ongoing work using cryoelectron microscopy of vitreous ice-embedded samples as a quantitative tool to investigate the assembly and organization of two important biological structures, namely, microtubules and viruses, in particular the bluetongue virus.

Animals↗
PubMed2001

Structures of unliganded and ATP-bound states of the Escherichia coli chaperonin GroEL by cryoelectron microscopy.

We have developed an angular refinement procedure incorporating correction for the microscope contrast transfer function, to determine cryoelectron microscopy (cryo-EM) structures of the Escherichia coli chaperonin GroEL in its apo and ATP-bound forms. This image reconstruction procedure is verified to 13-A resolution by comparison of the cryo-EM structure of unliganded GroEL with the crystal structure. Binding, encapsulation, and release of nonnative proteins by GroEL and its cochaperone GroES are controlled by the binding and hydrolysis of ATP. Seven ATP molecules bind cooperatively to one heptameric ring of GroEL. This binding causes long-range conformational changes that determine the orientations of remote substrate-binding sites, and it also determines the conformation of subunits in the opposite ring of GroEL, in a negatively cooperative mechanism. The conformation of GroEL-ATP was determined at approximately 15-A resolution. In one ring of GroEL-ATP, the apical (substrate-binding) domains are extremely disordered, consistent with the high mobility needed for them to achieve the 60 degrees elevation and 90 degrees twist of the GroES-bound state. Unexpectedly, ATP binding also increases the separation between the two rings, although the interring contacts are present in the density map.

Adenosine Triphosphate↗
PubMed1997

IRIS explorer software for radial-depth cueing reovirus particles and other macromolecular structures determined by cryoelectron microscopy and image reconstruction.

Structures of biological macromolecules determined by transmission cryoelectron microscopy (cryo-TEM) and three-dimensional image reconstruction are often displayed as surface-shaded representations with depth cueing along the viewed direction (Z cueing). Depth cueing to indicate distance from the center of virus particles (radial-depth cueing, or R cueing) has also been used. We have found that a style of R cueing in which color is applied in smooth or discontinuous gradients using the IRIS Explorer software is an informative technique for displaying the structures of virus particles solved by cryo-TEM and image reconstruction. To develop and test these methods, we used existing cryo-TEM reconstructions of mammalian reovirus particles. The newly applied visualization techniques allowed us to discern several new structural features, including sites in the inner capsid through which the viral mRNAs may be extruded after they are synthesized by the reovirus transcriptase complexes. To demonstrate the broad utility of the methods, we also applied them to cryo-TEM reconstructions of human rhinovirus, native and swollen forms of cowpea chlorotic mottle virus, truncated core of pyruvate dehydrogenase complex from Saccharomyces cerevisiae, and flagellar filament of Salmonella typhimurium. We conclude that R cueing with color gradients is a useful tool for displaying virus particles and other macromolecules analyzed by cryo-TEM and image reconstruction.

Animals↗
PubMedDate not supplied

Structure of bluetongue virus particles by cryoelectron microscopy.

The structure of the bluetongue virus (BTV) particle, determined by cryoelectron microscopy and image analysis, reveals a well-ordered outer shell which differs markedly from other known Reoviridae. The inner shell is known to have an icosahedral structure with 260 triangular spikes of VP7 trimers arranged on a T = 13,l lattice. The outer shell is seen to consist of 120 globular regions (possibly VP5), which sit neatly on each of the six-membered rings of VP7 trimers. "Sail"-shaped spikes located above 180 of the VP7 trimers form 60 triskelion-type motifs which cover all but 20 of the VP7 trimers. These spikes are possibly the hemagglutinating protein VP2 which contains a virus neutralization epitope. Thus, VP2 and VP5 together form a continuous layer around the inner shell except for holes on the 5-fold axis.

Bluetongue virus↗
PubMed1990

Ligand-dependent structural variations in Escherichia coli F1 ATPase revealed by cryoelectron microscopy.

The Escherichia coli F1 ATPase, ECF1, has been examined by cryoelectron microscopy after reaction with Fab' fragments generated from monoclonal antibodies to the alpha and epsilon subunits. The enzyme-antibody complexes appeared triangular due to the superposition of three anti-alpha Fab' fragments on alternating densities of the hexagonally arranged alpha and beta subunits. The Fab' to the epsilon subunit superimposed on a beta subunit. A density was observed near the center of the structure in the internal cavity. The position of this central density with respect to peripheral sites was not fixed. Sorting of images of ECF1 labeled with the combination of three anti-alpha Fab' fragments plus an Fab' directed to the epsilon subunit gave three classes in each of which the central density was closest to a different beta subunit. The distribution of the central density among the three classes was measured for different ligand-binding conditions. When ATP was present in catalytic sites under conditions where there was no enzyme turnover (i.e., without Mg2+ present), there were approximately equal numbers of images in each of three classes. When ATP and Mg2+ were added and ATP hydrolysis was allowed to proceed, almost two-thirds of the images were in the class in which the central density was closest to the beta subunit superimposed by the epsilon subunit. We conclude that domains within the ECF1 structure, either the central mass or a domain including the epsilon subunit, move in the enzyme in response to ligand binding. We suggest that this movement is involved in coupling catalytic sites to the proton channel in the F0 part of the ATP synthase.

Adenosine Diphosphate↗
PubMed1998

Three-dimensional reconstructions from cryoelectron microscopy images reveal an intimate complex between helicase DnaB and its loading partner DnaC.

BACKGROUND: DNA helicases play a fundamental role in all aspects of nucleic acid metabolism and defects in these enzymes have been implicated in a number of inherited human disorders. DnaB is the major replicative DNA helicase in Escherichia coli and has been used as a model system for studying the structure and function of hexameric helicases. The native protein is a hexamer of identical subunits, which in solution forms a complex with six molecules of the loading protein DnaC. DnaB is delivered from this complex onto the DNA template, with the subsequent release of DnaC. We report here the structures of the DnaB helicase hexamer and its complex with DnaC under a defined set of experimental conditions, as determined by three-dimensional cryoelectron microscopy. It was hoped that the structures would provide insight into the mechanisms of helicase activity. RESULTS: The DnaB structure reveals that six DnaB monomers assemble as three asymmetric dimers to form a polar, ring-like hexamer. The hexamer has two faces, one displaying threefold and the other sixfold symmetry. The six DnaC protomers bind tightly to the sixfold face of the DnaB hexamer. This is the first report of a three-dimensional structure of a helicase obtained using cryoelectron microscopy, and the first report of the structure of a helicase in complex with a loading protein. CONCLUSIONS: The structures of the DnaB helicase and its complex with DnaC reveal some interesting structural features relevant to helicase function and to the assembly of the two-protein complex. The results presented here provide a basis for a more complete understanding of the structure and function of these important proteins.

Bacterial Proteins↗
PubMed2005

Cryoelectron microscopy of liposomes.

A thin aqueous film of suspended lipid vesicles?micelles is the object of choice for vitrification and subsequent study by cryoelectron microscopy. Just prior to vitrification, a thin film (compare with a soap film) is vulnerable to heat and mass exchange. Preparation of thin films in a temperature- and humidity-controlled environment is essential to prevent osmotic and temperature-induced alterations of the lipid structure, as will be explained in this chapter. Further automation of the preparative procedure by automatic blotting and PC control over the timing of critical steps (including vitrification) may further assist in the reproducible throughput of high-quality specimens. By cryotomography, taking a tilt series under low-dose conditions, a three-dimensional reconstruction of the specimen can be analyzed.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1990

Three-dimensional structure of rhesus rotavirus by cryoelectron microscopy and image reconstruction.

The structure of rhesus rotavirus was examined by cryoelectron microscopy and image analysis. Three-dimensional reconstructions of infectious virions were computed at 26- and 37-A resolution from electron micrographs recorded at two different levels of defocus. The major features revealed by the reconstructions are (a) both outer and inner capsids are constructed with T = 13l icosahedral lattice symmetry; (b) 60 spikelike projections, attributed to VP4, extend at least 100 A from the outer capsid surface; (c) the outer capsid, attributed primarily to VP7, has a smoothly rippled surface at a mean radius of 377 A and is perforated by 132 aqueous holes ranging from 40-65 A in diameter; (d) the inner capsid has a "bristled" outer surface composed of 260 trimeric-shaped columns of density, attributed to VP6, which merge with a smooth, spherical shell of density at a lower, mean radius of 299 A, and which is perforated by holes in register with those in the outer capsid; (e) a "core" region contains a third, nonspherical shell of density at a mean radius of 225 A that encapsidates the double-stranded RNA genome; and (f) the space between the outer and inner capsids forms an open aqueous network that may provide pathways for the diffusion of ions and small regulatory molecules as well as the extrusion of RNA. The assignment of different viral structural proteins to specific features of the reconstruction has been tentatively made on the basis of excluded volume estimates and previous biochemical characterizations of rotavirus.

Animals↗
PubMed2006

Visualization of alpha-helices in a 6-angstrom resolution cryoelectron microscopy structure of adenovirus allows refinement of capsid protein assignments.

The structure of adenovirus was determined to a resolution of 6 A by cryoelectron microscopy (cryoEM) single-particle image reconstruction. Docking of the hexon and penton base crystal structures into the cryoEM density established that alpha-helices of 10 or more residues are resolved as rods. A difference map was calculated by subtracting a pseudoatomic capsid from the cryoEM reconstruction. The resulting density was analyzed in terms of observed alpha-helices and secondary structure predictions for the additional capsid proteins that currently lack atomic resolution structures (proteins IIIa, VI, VIII, and IX). Protein IIIa, which is predicted to be highly alpha-helical, is assigned to a cluster of helices observed below the penton base on the inner capsid surface. Protein VI is present in approximately 1.5 copies per hexon trimer and is predicted to have two long alpha-helices, one of which appears to lie inside the hexon cavity. Protein VIII is cleaved by the adenovirus protease into two fragments of 7.6 and 12.1 kDa, and the larger fragment is predicted to have one long alpha-helix, in agreement with the observed density for protein VIII on the inner capsid surface. Protein IX is predicted to have one long alpha-helix, which also has a strongly indicated propensity for coiled-coil formation. A region of density near the facet edge is now resolved as a four-helix bundle and is assigned to four copies of the C-terminal alpha-helix from protein IX.

Adenoviridae↗
PubMed2000

Structure of a molluscan hemocyanin didecamer (HtH1 from Haliotis tuberculata) at 12 A resolution by cryoelectron microscopy.

A 12 A resolution three-dimensional density map of the Haliotis tuberculata hemocyanin type 1 (HtH1) didecamer has been obtained by cryoelectron microscopy of unstained molecules and angular reconstitution. The dyad symmetry of the 8 MDa D5 HtH1 didecamer, formed by the pairing of two asymmetric 4 MDa ring-like C5 decamers, is emphasised. The major and minor surface helical grooves of the didecamer are well defined, in agreement with earlier data on molluscan hemocyanins. The location of the obliquely orientated repeating unit, a subunit dimer, within the decamer has been defined. Following interactive extraction of this dimer, several new structural features of the dimer and of the subunit have now emerged with improved detail. The subunit dimer possesses pseudo 2-fold symmetry, resulting from the steric arrangement of the wall domains/functional units (FUs-abcdef) of the two subunits. The arc and collar FUs (g and h) depart from this inherent 2-fold symmetry and are thereby responsible for the asymmetry of the C5 decamer, with the internalised collar/arc complex at one edge of the decamer. The FU heterodimers forming the wall morphological units have a hollow centre, and thus create a series of repeating channels that extend within the wall through all three tiers of the decamer. The connections between the wall and the arc are defined with improved clarity, and evidence is provided to indicate that the arc and collar FU pairs have a homodimeric composition (gg and hh, respectively). Two possibilities for the subunit path within the subunit dimer are presented, which correlate with the available structural, immunolabelling and protease cleavage data from HtH1 and other molluscan hemocyanins.

Animals↗
PubMed2001

Cryoelectron microscopy of lambda phage DNA condensates in vitreous ice: the fine structure of DNA toroids.

DNA toroids produced by the condensation of lambda phage DNA with hexammine cobalt (III) have been investigated by cryoelectron microscopy. Image resolution obtained by this technique has allowed unprecedented views of DNA packing within toroidal condensates. Toroids oriented coplanar with the microscope image plane exhibit circular fringes with a repeat spacing of 2.4 nm. For some toroids these fringes are observed around almost the entire circumference of the toroid. However, for most toroids well-defined fringes are limited to less than one-third of the total toroid circumference. Some toroids oriented perpendicular to the image plane reveal DNA polymers organized in a hexagonal close-packed lattice; however, for other toroids alternative packing arrangements are observed. To aid interpretation of electron micrographs, three-dimensional model toroids were generated with perfect hexagonal DNA packing throughout, as well as more physically realistic models that contain crossover points between DNA loops. Simulated transmission electron microscopy images of these model toroids in different orientations faithfully reproduce most features observed in cryoelectron micrographs of actual toroids.

Bacteriophage lambda↗
PubMed2001

The three-dimensional structure of alpha-actinin obtained by cryoelectron microscopy suggests a model for Ca(2+)-dependent actin binding.

The three-dimensional structure of alpha-actinin from rabbit skeletal muscle was determined by cryoelectron microscopy in combination with homology modeling of the separate domain structures based on results previously determined by X-ray crystallography and nuclear magnetic resonance spectroscopy. alpha-Actinin was induced to form two-dimensional arrays on a positively charged lipid monolayer and micrographs were collected from unstained, frozen hydrated specimens at tilt angles from 0 degrees to 60 degrees. Interpretation of the 15 A-resolution three-dimensional structure was done by manually docking homologous models of the three key domains, actin-binding, three-helix motif and the C-terminal calmodulin-like domains. The initial model was refined quantitatively to improve its fit to the experimental reconstruction. The molecular model of alpha-actinin provides the first view of the overall structure of a complete actin cross-linking protein. The structure is characterized by close proximity of the C-terminal, calmodulin-like domain to the linker between the two calponin-homology domains that comprise the actin-binding domain. This location suggests a hypothesis to explain the involvement of the C-terminal domain in Ca(2+)-dependent actin binding of non-muscle isoforms.

Actinin↗
PubMed1999

Effect of buffer conditions on the position of tRNA on the 70 S ribosome as visualized by cryoelectron microscopy.

The effect of buffer conditions on the binding position of tRNA on the Escherichia coli 70 S ribosome have been studied by means of three-dimensional (3D) cryoelectron microscopy. Either deacylated tRNAfMet or fMet-tRNAfMet were bound to the 70 S ribosomes, which were programmed with a 46-nucleotide mRNA having AUG codon in the middle, under two different buffer conditions (conventional buffer: containing Tris and higher Mg2+ concentration [10-15 mM]; and polyamine buffer: containing Hepes, lower Mg2+ concentration [6 mM], and polyamines). Difference maps, obtained by subtracting 3D maps of naked control ribosome in the corresponding buffer from the 3D maps of tRNA.ribosome complexes, reveal the distinct locations of tRNA on the ribosome. The position of deacylated tRNAfMet depends on the buffer condition used, whereas that of fMet-tRNAfMet remains the same in both buffer conditions. The acylated tRNA binds in the classical P site, whereas deacylated tRNA binds mostly in an intermediate P/E position under the conventional buffer condition and mostly in the position corresponding to the classical P site, i. e. in the P/P state, under the polyamine buffer conditions.

Acylation↗
PubMed2000

3D map of the plant photosystem II supercomplex obtained by cryoelectron microscopy and single particle analysis.

Here we describe the first 3D structure of the photosystem II (PSII) supercomplex of higher plants, constructed by single particle analysis of images obtained by cryoelectron microscopy. This large multisubunit membrane protein complex functions to absorb light energy and catalyze the oxidation of water and reduction of plastoquinone. The resolution of the 3D structure is 24 A and emphasizes the dimeric nature of the supercomplex. The extrinsic proteins of the oxygen-evolving complex (OEC) are readily observed as a tetrameric cluster bound to the lumenal surface. By considering higher resolution data, obtained from electron crystallography, it has been possible to relate the binding sites of the OEC proteins with the underlying intrinsic membrane subunits of the photochemical reaction center core. The model suggests that the 33 kDa OEC protein is located towards the CP47/D2 side of the reaction center but is also positioned over the C-terminal helices of the D1 protein including its CD lumenal loop. In contrast, the model predicts that the 23/17 kDa OEC proteins are positioned at the N-terminus of the D1 protein incorporating the AB lumenal loop of this protein and two other unidentified transmembrane helices. Overall the 3D model represents a significant step forward in revealing the structure of the photosynthetic OEC whose activity is required to sustain the aerobic atmosphere on our planet.

Cryoelectron Microscopy↗