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At least 109 records · Page 6Linked to original sources
PubMed2001

Cryoelectron-microscopy image reconstruction of symmetry mismatches in bacteriophage phi29.

A method has been developed for three-dimensional image reconstruction of symmetry-mismatched components in tailed phages. Although the method described here addresses the specific case where differing symmetry axes are coincident, the method is more generally applicable, for instance, to the reconstruction of images of viral particles that deviate from icosahedral symmetry. Particles are initially oriented according to their dominant symmetry, thus reducing the search space for determining the orientation of the less dominant, symmetry-mismatched component. This procedure produced an improved reconstruction of the sixfold-symmetric tail assembly that is attached to the fivefold-symmetric prolate head of phi29, demonstrating that this method is capable of detecting and reconstructing an object that included a symmetry mismatch. A reconstruction of phi29 prohead particles using the methods described here establishes that the pRNA molecule has fivefold symmetry when attached to the prohead, consistent with its proposed role as a component of the stator in the phi29 DNA packaging motor.

Bacillus Phages↗
PubMed1999

Cryoelectron microscopy of a nucleating model bile in vitreous ice: formation of primordial vesicles.

Because gallstones form so frequently in human bile, pathophysiologically relevant supersaturated model biles are commonly employed to study cholesterol crystal formation. We used cryo-transmission electron microscopy, complemented by polarizing light microscopy, to investigate early stages of cholesterol nucleation in model bile. In the system studied, the proposed microscopic sequence involves the evolution of small unilamellar to multilamellar vesicles to lamellar liquid crystals and finally to cholesterol crystals. Small aliquots of a concentrated (total lipid concentration = 29.2 g/dl) model bile containing 8.5% cholesterol, 22.9% egg yolk lecithin, and 68.6% taurocholate (all mole %) were vitrified at 2 min to 20 days after fourfold dilution to induce supersaturation. Mixed micelles together with a category of vesicles denoted primordial, small unilamellar vesicles of two distinct morphologies (sphere/ellipsoid and cylinder/arachoid), large unilamellar vesicles, multilamellar vesicles, and cholesterol monohydrate crystals were imaged. No evidence of aggregation/fusion of small unilamellar vesicles to form multilamellar vesicles was detected. Low numbers of multilamellar vesicles were present, some of which were sufficiently large to be identified as liquid crystals by polarizing light microscopy. Dimensions, surface areas, and volumes of spherical/ellipsoidal and cylindrical/arachoidal vesicles were quantified. Early stages in the separation of vesicles from micelles, referred to as primordial vesicles, were imaged 23-31 min after dilution. Observed structures such as enlarged micelles in primordial vesicle interiors, segments of bilayer, and faceted edges at primordial vesicle peripheries are probably early stages of small unilamellar vesicle assembly. A decrease in the mean surface area of spherical/ellipsoidal vesicles was correlated with the increased production of cholesterol crystals at 10-20 days after supersaturation by dilution, supporting the role of small unilamellar vesicles as key players in cholesterol nucleation and as cholesterol donors to crystals. This is the first visualization of an intermediate structure that has been temporally linked to the development of small unilamellar vesicles in the separation of vesicles from micelles in a model bile and suggests a time-resolved system for further investigation.

Bile↗
PubMed2005

Cryoelectron microscopy reveals new features in the three-dimensional structure of phosphorylase kinase.

Phosphorylase kinase (PhK), a regulatory enzyme in the cascade activation of glycogenolysis, is a 1.3-MDa hexadecameric complex, (alphabetagammadelta)(4). PhK comprises two arched octameric (alphabetagammadelta)(2) lobes that are oriented back-to-back with overall D(2) symmetry and connected by small bridges. These interlobal bridges, arguably the most questionable structural component of PhK, are one of several structural features that potentially are artifactually generated or altered by conventional sample preparation techniques for electron microscopy (EM). To minimize such artifacts, we have solved by cryoEM the first three-dimensional (3D) structure of nonactivated PhK from images of frozen hydrated molecules of the kinase. Minimal dose electron micrographs of PhK in vitreous ice revealed particles in a multitude of orientations. A simple model was used to orient the individual images for 3D reconstruction, followed by multiple rounds of refinement. Three-dimensional reconstruction of nonactivated PhK from approximately 5000 particles revealed a bridged, bilobal molecule with a resolution estimated by Fourier shell correlation analysis at 25 A. This new structure suggests that several prominent features observed in the structure of PhK derived from negatively stained particles arise as artifacts of specimen preparation. In comparison to the structure from negative staining, the cryoEM structure shows three important differences: (1) a dihedral angle between the two lobes of approximately 90 degrees instead of 68 degrees, (2) a compact rather than extended structure for the lobes, and (3) the presence of four, rather than two, connecting bridges, which provides the first direct evidence for these components as authentic elements of the kinase solution structure.

Animals↗
PubMed2003

Revisiting the structure of Alvinella pompejana hemoglobin at 20A resolution by cryoelectron microscopy.

The hemoglobin of the polychaete worm Alvinella pompejana was reconstructed at 20A resolution from frozen-hydrated samples observed by electron microscopy according to the random conical tilt series method. This three-dimensional reconstruction was mirror-inverted with respect to a previous volume published by de Haas et al. in 1996. In order to explain this handedness discrepancy, various 3D reconstructions using different reference volumes were carried out showing that the choice of the first volume was the keystone during the refinement process. The 3D reconstruction volume of A. pompejana Hb presented structural features characteristic of annelid Hbs with two hexagonal layers each comprising six hollow globular subassemblies and a complex of non-heme linker chains. Moreover, the eclipsed conformation of the two hexagonal layers and a HGS architecture similar to that described for Arenicola marina Hb led to the conclusion that A. pompejana Hb belonged to the architectural type II according to the definition of Jouan et al. (2001). A comparison between this cryo-EM volume and X-ray crystallography density maps of Lumbricus terrestris type-I Hb (Royer et al., 2000) showed that the triple stranded coiled coil structures of linker chains were different. Based on this observation, a model was proposed to explain the eclipsed conformation of the two hexagonal layers of type-II Hbs.

Animals↗
PubMed2001

The organization of mature Rous sarcoma virus as studied by cryoelectron microscopy.

We have studied the organization of mature infectious Rous sarcoma virus (RSV), suspended in vitreous ice, using transmission electron microscopy. The enveloped virions are spherical in shape, have a mean diameter of 127 nm, and vary significantly in size. Image processing reveals the presence of the viral matrix protein underlying the lipid bilayer and the viral envelope proteins external to the lipid bilayer. In the interior of the virus, the characteristic mature retroviral core is clearly imaged. In contrast to lentiviruses, such as human immunodeficiency virus, the core of RSV is essentially isometric. The capsid, or external shell of the core, has a faceted, almost polygonal appearance in electron micrographs, but many capsids also exhibit continuous surface curvature. Cores are not uniform in size or shape. Serrations observed along the projected faces of the core suggest a repetitive molecular structure. Some isolated cores were observed in the sample, confirming that cores are at least transiently stable in the absence of the viral envelope. Using an approach grounded in geometric probability, we estimate the size of the viral core from the projection data. We show that the size of the core is not tightly controlled and that core size and virion size are positively correlated. From estimates of RNA packing density we conclude that either the RNA within the core is loosely packed or, more probably, that it does not fill the core.

Animals↗
PubMed1995

Putative receptor binding sites on alphaviruses as visualized by cryoelectron microscopy.

The structures of Sindbis virus and Ross River virus complexed with Fab fragments from monoclonal antibodies have been determined from cryoelectron micrographs. Both antibodies chosen for this study bind to regions of the virions that have been implicated in cell-receptor recognition and recognize epitopes on the E2 glycoprotein. The two structures show that the Fab fragments bind to the outermost tip of the trimeric envelope spike protein. Hence, the same region of both the Sindbis virus and Ross River virus envelope spike is composed of E2 and is involved in recognition of the cellular receptor.

Antibodies, Monoclonal↗
PubMed1996

Three-dimensional cryoelectron microscopy of dimeric kinesin and ncd motor domains on microtubules.

Kinesin and ncd motor proteins are homologous in sequence yet move in opposite directions along microtubules. We have previously shown that monomeric kinesin and ncd bind in the same orientation on equivalent sites relative to the ends of tubulin sheets of known polarity. We now report cryoelectron microscope images of 16-protofilament microtubules decorated with both single- and double-headed kinesin and double-headed ncd. Three-dimensional density maps and difference maps show that, in adenosine 5'-[beta,gamma-imido]triphosphate, both dimeric motors bind tightly to microtubules via one head, leaving the other free, though apparently in a fixed position. The attached heads of dimers bind to tubulin in the same way as single kinesin heads. The second heads are connected to the tops of the first but, whereas the second kinesin head is closely associated with the first, pairs of ncd heads are splayed apart. There is also a distinct difference in orientation: the second kinesin head is tilted toward the microtubule plus end, while the second head of ncd points toward the minus end.

Adenosine Diphosphate↗
PubMed1996

Three-dimensional reconstruction of the hexagonal bilayer hemoglobin of the hydrothermal vent tube worm Riftia pachyptila by cryoelectron microscopy.

A frozen-hydrated specimen of the V1 hemoglobin of the hydrothermal vent tube worm Riftia pachyptila was observed in the electron microscope and subjected to three-dimensional reconstruction by the method of random conical tilt series. The 3D volume possesses a D6 point-group symmetry. When viewed along its 6-fold axis the vertices of its upper hexagonal layer are 16 degrees clockwise rotated compared to those of the lower layer. A central linker complex is decorated by 12 hollow globular substructures. The linker complex comprises (i) a central hexagonal toroid, (ii) two internal bracelets onto which the hollow globular substructures are built, and (iii) six structures connecting the two hexagonal layers. The hollow globular substructures, related to the dodecamers of globin chains resulting from the dissociation of the hexagonal bilayer hemoglobin, have a local pseudo 3-fold symmetry and are composed each of three elongated structures visible when the volume is displayed at high threshold. At a resolution of 36 A, the 3D volumes of the hexagonal bilayer hemoglobins of Riftia pachyptyla and of the leech Macrobdella decora look almost perfectly identical.

Animals↗
PubMed1996

Three-dimensional reconstruction by cryoelectron microscopy of the giant hemoglobin of the polychaete worm Alvinella pompejana.

A frozen-hydrated specimen of the hexagonal bilayer hemoglobin (HBL Hb) from the deep-sea hydrothermal vent polychaete worm Alvinella pompejana, the most thermophilic metazoan known to date, was observed in the electron microscope and subjected to three-dimensional (3D) reconstruction by the method of random conical tilt series. At a resolution of 34.6 A by the differential phase residual method and 27.7 A by the Fourier shell correlation method, the 3D volume possesses a D6 point-group symmetry. While in previous 3D reconstructions of annelid and vestimentiferan Hbs the vertices of the upper layer were 16 degrees rotated compared with those of the lower layer, in Alvinella Hb the vertices of the two hexagonal layers are almost perfectly eclipsed when viewed along the 6-fold axis. As in the HBL Hbs of Riftia pachyptila and Macrobdella decora, a central linker complex is decorated by 12 hollow globular substructures (HGS). The linker complex comprises (1) a central hexagonal toroid, (2) two internal bracelets onto which the HGSs are built, and (3) six connections between the two hexagonal layers. Each HGS is composed of six masses, which are separated when the volume is displayed at high threshold, plus one additional mass involved in the bracelet connecting the six HGSs in both hexagonal layers. The HGSs have a local pseudo 3-fold symmetry and a disposition of the high-density masses different from those of Riftia V1 Hb.

Animals↗
PubMed1997

Three-dimensional reconstruction of Limulus polyphemus hemocyanin from cryoelectron microscopy.

Hemocyanin (Hc) the respiratory pigment of the horseshoe crab Limulus polyphemus (Lp) is composed of 48 approximately 75 kDa copper-containing subunits arranged in eight hexameric groups. In this study, we used the random conical tilt series method to do a three-dimensional (3D) reconstruction of Lp Hc observed in vitreous ice. This approach allowed the unambiguous determination of the handedness of the molecule. Lp Hc contains two superimposed 4 x 6mer structures possessing the same structural features as the other 4 x 6meric Hcs, namely flip and flop views and a rocking effect. Moreover, 3D fitting of the X-ray structure of subunit LpII with the reconstruction volume shows that the intra4 x 6meric contacts described in arthropod Hcs also occur within Limulus Hc. The two half-molecules composing the 8 x 6mer have their flop faces in contact (flop/flop association), the main links being formed by subunits LpIV. Model building shows that the flop/flop association is the only possible arrangement which allows the assembly of the whole particle. The two alternate constructions (flip/flop and flip/flip) are forbidden because of steric hindrance.

Algorithms↗
PubMed1997

Three-dimensional cryoelectron microscopy of 16-protofilament microtubules: structure, polarity, and interaction with motor proteins.

We present a three-dimensional (3D) map, reconstructed from electron microscope (EM) images of naturally occurring 16-protofilament (PF) microtubules (MTs) in ice. We compare it with the tubulin in six 3D maps of MTs decorated with motor domains, three from frozen MTs decorated with kinesin or ncd in the tightly bound AMP-PNP state, and three from negatively stained MTs decorated with kinesin in different nucleotide states. The comparison confirms that kinesin and ncd bind to identical sites and interact with both monomers of a tubulin dimer. Maps of specimens in negative stain and in ice are similar except that the protein in the top half of a motor domain appears denser in negative stain. The interactions have only a small effect on tubulin structure; the outward appearance is unchanged, but there seems to be a small internal rearrangement. The relative polarity of undecorated and decorated MTs is evident from their 3D structures. This agrees with the absolute polarities indicated by the orientations of motors in decorated specimens and by polar superposition patterns calculated for undecorated MTs. An image of tubulin PFs in zinc-induced sheets has been tentatively oriented by similar criteria.

Adenylyl Imidodiphosphate↗
PubMed1994

Direct imaging of interactions between an icosahedral virus and conjugate F(ab) fragments by cryoelectron microscopy and X-ray crystallography.

The binding properties of seven mouse monoclonal antibodies (McAbs) raised against cowpea mosaic virus (CPMV) were characterized by conventional and inhibition enzyme-linked immunosorbent assay (ELISA) technique. McAb binding to CPMV on electron microscope (EM) grids was also assayed with gold-labeled anti-mouse antibodies. Two of the seven McAbs (5B2 and 10B7) were found to bind tighter to CPMV than the others in the inhibition ELISA and the EM assay. F(ab) fragments from both of these McAbs were prepared and complexed with CPMV in solution. Electron micrographs of flash frozen (vitrified) samples of native CPMV and CPMV complexed with F(ab) fragments from McAbs 5B2 and 10B7 as well as IgGs from 5B2 were recorded and reconstructions were computed at 23 A resolution for the CPMV/F(ab) complexes and 30 A resolution for the CPMV/IgG complex. Structures of all three complexes clearly displayed the F(ab) fragments distributed with icosahedral symmetry on the surface of CPMV. The IgG bound in a monodentate fashion with only one F(ab) attached to the virus surface. F(ab) fragments from 5B2 and 10B7 bound to nearly identical positions. The refined 2.8 A X-ray structure of CPMV was used to identify the roughly 30 amino acids covered by the F(ab) fragments. The "footprint" spans a subunit interface and appears spatially similar to antigenic site 3B on poliovirus. In a previous, preliminary report of the CPMV/F(ab) 5B2 complex (Wang et al., 1992, Nature 355, 275-278) the wrong enantiomorph of the reconstruction was chosen. This was corrected and, since the F(ab) binds close to a mirror plane, the change in the footprint was minor.

Animals↗
PubMed1988

Cryoelectron microscopy of vitrified Sendai virions.

Morphology of vitrified Sendai virions was studied by transmission type electron microscopy. Almost all the virions appeared to be completely spherical, although their diameters differed. A possibly continuously long nucleocapsid was seen running helically in an envelope. Spikes were seen on the virion surfaces. The results indicate pleomorphism of Sendai virions in size but not in shape.

Freezing↗
PubMed1991

Structural studies of human alpha 2-macroglobulin: concordance between projected views obtained by negative-stain and cryoelectron microscopy.

Two views of native alpha 2-macroglobulin are revealed by electron microscopy of negatively stained samples; in one view the molecule resembles a padlock and in the other, a pair of lips. Interconversion of the two views upon tilting establishes that these are two different projected views of the same structure. Furthermore, the two views are related by a 45 degrees rotation about their major axis because they interconvert when the specimens are titled +/- 22.5 degrees. Negatively stained molecules on Butvar films present a nearly equal distribution of the two views, whereas in frozen-hydrated samples the molecules almost exclusively are oriented in the lip view. Measurements from both views indicate that the alpha 2-macroglobulin molecule is approximately 200 A long and approximately 140 A wide. Our results suggest that alpha 2-macroglobulin is composed of two protomeric units, each in the shape of a twisted letter S. These units are joined together at their ends to form a complex with point group symmetry 222. The 45 degrees interconversion angle between the lip and padlock views support this arrangement. Average images of unstained and stained lips are quite similar, indicating that the native structure is consistently preserved by the two electron microscopy procedures used in this investigation. This is substantiated by the interconversion between the lip and padlock views that occurs when the molecule is rotated 45 degrees [corrected] about its major twofold axis.

Cryopreservation↗
PubMed1995

Cryoelectron microscopy of low density lipoprotein in vitreous ice.

In this report, images of low density lipoprotein (LDL) in vitreous ice at approximately 30 A resolution are presented. These images show that LDL is a quasi-spherical particle, approximately 220-240 A in diameter, with a region of low density (lipid) surrounded by a ring (in projection) of high density believed to represent apolipoprotein B-100. This ring is seen to be composed of four or five (depending on view) large regions of high density material that may represent protein superdomains. Analysis of LDL images obtained at slightly higher magnification reveals that areas of somewhat lower density connect these regions, in some cases crossing the projectional interiors of the LDL particles. Preliminary image analysis of LDL covalently labeled at Cys3734 and Cys4190 with 1.4-nm Nanogold clusters demonstrates that this methodology will provide an important site-specific marker in studies designed to map the organization of apoB at the surface of LDL.

Centrifugation, Density Gradient↗