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At least 235 records · Page 13Linked to original sources

PubMed1991

Time-resolved cryo-electron microscopic study of the dissociation of actomyosin induced by photolysis of photolabile nucleotides.

The rapid release of a substrate or other ligand from photolabile precursors in a thin layer suspension of biological specimens followed by rapid freezing provides a method of trapping and visualizing short-lived states in a dynamic system. We demonstrate here the first successful application of this method to study the interaction of actin filaments with myosin subfragment 1 (S1) after release of nucleotides. The results obtained suggest that structural changes in actin filaments occur as a result of interaction with S1.

Actins↗

PubMed1993

Capsid localization of the bacteriophage P4 Psu protein.

In addition to its polarity-suppressing activity, the Psu protein of bacteriophage P4 also serves to stabilize the capsid against heat treatment and binds externally to the phage capsid. However, the heat stability is lost upon purification of the virus, indicating a loss of Psu protein from the capsid. By using three-dimensional reconstruction from cryo-electron micrographs of P4 psu1 amber mutants lacking Psu, and of P4 virions, which have been saturated with Psu protein to regain heat stability, we have determined the position of this protein on the virus surface. Our results are consistent with the hypothesis that the function of Psu is to stabilize the hexameric capsomer assembly.

Capsid↗

PubMed1998

Cryo-electron diffraction as a tool to study local variations in the lipid organization of human stratum corneum.

The human skin provides the body with a barrier against transepidermal water loss and the penetration of harmful agents (e.g. microbes) from outside. This barrier function is produced mainly by the outermost, nonviable layer of the epidermis, the stratum corneum (s.c.). The s.c. consists of terminally differentiated corneocytes surrounded by a continuous intercellular lipid domain, which contains mostly ceramides, cholesterol and free fatty acids. Small- and wide-angle X-ray diffraction studies have elucidated the lamellar and lateral lipid organizations in these domains. However, these techniques require bulk quantities of SC, as a result of which local structure information on the lipids cannot be obtained. Insights to these local lipid arrangements are important when new transdermal drug delivery systems have to be developed. Therefore, the technique of electron diffraction arose as a tool to study the lateral packing of the lipids in the intercellular domains of SC, locally. In a previous study, the suitability of electron diffraction was demonstrated using a lipid model system that resembled the lipid composition of the SC. The spacings calculated from the electron diffraction patterns were in good agreement with the spacings revealed by wide-angle X-ray diffraction. The results presented here succeed this previous study. We improved the microscope settings and developed a new preparation method to study ex vivo human s.c. by cryo-electron diffraction. The method is based on the conventional tape-stripping method and offers the possibility to study depth-related changes in the lipid organization of human SC. Diffraction patterns of both hexagonal and orthorhombic lipid lattices have been recorded with spacings that resembled those found in human s.c. by wide-angle X-ray diffraction. After lipid extraction, such diffraction patterns could no longer be detected in the samples.

Epidermis↗

PubMed1992

Magnification mismatches between micrographs: corrective procedures and implications for structural analysis.

Quantitative structural analysis from electron micrographs of biological macromolecules inevitably requires the synthesis of data from many parts of the same micrograph and, ultimately, from multiple micrographs. Higher resolutions require the inclusion of progressively more data, and for the particles analyzed to be consistent to within ever more stringent limits. Disparities in magnification between micrographs or even within the field of one micrograph, arising from lens hysteresis or distortions, limit the resolution of such analyses. A quantitative assessment of this effect shows that its severity depends on the size of the particle under study: for particles that are 100 nm in diameter, for example, a 2% discrepancy in magnification restricts the resolution to approximately 5 nm. In this study, we derive and describe the properties of a family of algorithms designed for cross-calibrating the magnifications of particles from different micrographs, or from widely differing parts of the same micrograph. This approach is based on the assumption that all of the particles are of identical size: thus, it is applicable primarily to cryo-electron micrographs in which native dimensions are precisely preserved. As applied to icosahedral virus capsids, this procedure is accurate to within 0.1-0.2%, provided that at least five randomly oriented particles are included in the calculation. The algorithm is stable in the presence of noise levels typical of those encountered in practice, and is readily adaptable to non-isometric particles. It may also be used to discriminate subpopulations of subtly different sizes.

Algorithms↗

PubMed1995

Proposal of alignment-independent classification of electron microscopic images with helical symmetry and its application to reconstituted thin filaments os skeletal muscle.

A new method to classify noisy images of filaments with helical symmetry was developed and applied to cryo-electron microscopic images of skeletal muscle thin filaments. In this method, images are transformed to minimum data sets which are independent of the relative view parameters, such as rotation of a three-dimensional object around the axis of helical symmetry, translation along an axis and operation to make a filament upside down, and classified by a principal component analysis. By this method, atypical data can be identified without aligning procedures, and small structural changes and polymorphic variation are expected to be detected objectively.

Actin Cytoskeleton↗

PubMed1997

Two-dimensional crystallization of the light-harvesting I-reaction centre photounit from Rhodospirillum rubrum.

A stoichiometric unit of the light-harvesting complex I and the reaction centre (LHI-RC complex) has been isolated from a carotenoid-less mutant of the purple non-sulphur bacterium Rhodospirillum rubrum by mild solubilization of photosynthetic membranes with the phospholipid detergent diheptylphosphatidylcholine. Dialysis of the isolated LHI-RC complexes in the presence of added dioleoyl-sn-phosphatidylcholine produced ordered two-dimensional crystals. Digital image processing revealed that the LHI-RC are packed together in a square lattice (a = b = 16.3 nm). The dimensions of the LHI ring are essentially identical with those determined from two-dimensional (2D) crystals of purified carotenoid-containing light-harvesting I complexes after analysis by cryo-electron microscopic techniques or from negatively stained 2D crystals of purified LHI complexes from a carotenoid-less mutant. Each LHI ring of the LHI-RC complex contains a central diffuse stain-excluding region, which is assigned to the reaction centre. The analysis of the LHI-RC 2D crystals strongly suggests that the geometry and subunit stoichiometry of the LHI ring is unaffected by the presence of a reaction centre that can probably assume various orientations within the ring.

Bacterial Proteins↗

PubMed1997

Novel structural features of bovine papillomavirus capsid revealed by a three-dimensional reconstruction to 9 A resolution.

The three-dimensional structure of bovine papillomavirus has been determined to 9 A resolution by reconstruction of high resolution, low dose cryo-electron micrographs of quench-frozen virions. Although hexavalent and pentavalent capsomeres form star-shaped pentamers of the major capsid protein L1, they have distinct high-resolution structures. Most prominently, a 25 A hole in the centre of hexavalent capsomeres is occluded in the pentavalent capsomeres. This raises the possibility that the L2 minor capsid protein is located in the centre of the pentavalent capsomeres. Inter-capsomere connections approximately 10 A in diameter were clearly resolved. These link adjacent capsomeres and are reminiscent of the helical connections that stabilize polyomavirus.

Animals↗

PubMed1989

Three-dimensional structure of the HSV1 nucleocapsid.

The three-dimensional structures of full and empty capsids of HSV1 were determined by computer analysis of low dose cryo-electron images of ice embedded capsids. The full capsid structure is organized into outer, intermediate, and inner structural layers. The empty capsid structure has only one layer which is indistinguishable from the outer layer of the full capsids. This layer is arranged according to T = 16 icosahedral symmetry. The intermediate layer of full capsids appears to lie on a T = 4 icosahedral lattice. The genomic DNA is located inside the T = 4 shell and is the component of the innermost layer of the full capsids. The outer and intermediate layers interact in such a way that the channels along their icosahedral two-fold axis coincide and form a direct pathway between the DNA and the environment outside the capsid.

Capsid↗

PubMed1991

Image reconstruction reveals the complex molecular organization of adenovirus.

The three-dimensional structure of adenovirus has been determined by image reconstruction from cryo-electron micrographs. Comparison with the high resolution X-ray crystal structure of hexon, the major capsid protein, enabled an unusually detailed interpretation of the density map and confirmed the validity of the reconstruction. The hexon packing in the capsid shows more extensive intermolecular interfaces between facets than previously proposed. The reconstruction provides the first three-dimensional visualization of the vertex proteins, including the penton base and its associated protruding fiber. Three minor capsid proteins that stabilize and modulate capsomer interactions are revealed. One of these components stabilizes the group-of-nine hexons in the center of each facet and the other two bridge hexons in adjacent facets. The strategic positions of these proteins highlight the importance of cementing proteins in stabilizing a complex assembly.

Adenoviruses, Human↗

PubMed1994

Finding a needle in a haystack: detection of a small protein (the 12-kDa VP26) in a large complex (the 200-MDa capsid of herpes simplex virus).

Macromolecular complexes that consist of homopolymeric protein frameworks with additional proteins attached at strategic sites for a variety of structural and functional purposes are widespread in subcellular biology. One such complex is the capsid of herpes simplex virus type 1 whose basic framework consists of 960 copies of the viral protein, VP5 (149 kDa), arranged in an icosahedrally symmetric shell. This shell also contains major amounts of three other proteins, including VP26 (12 kDa), a small protein that is approximately equimolar with VP5 and accounts for approximately 6% of the capsid mass. With a view to inferring the role of VP26 in capsid assembly, we have localized it by quantitative difference imaging based on three-dimensional reconstructions calculated from cryo-electron micrographs. Purified capsids from which VP26 had been removed in vitro by treatment with guanidine hydrochloride were compared with preparations of the same depleted capsids to which purified VP26 had been rebound and with native (undepleted) capsids. The resulting three-dimensional density maps indicate that six VP26 subunits are distributed symmetrically around the outer tip of each hexon protrusion on VP26-containing capsids. Because VP26 may be readily dissociated from and reattached to the capsid, it does not appear to contribute significantly to structural stabilization. Rather, its exposed location suggests that VP26 may be involved in linking the capsid to the surrounding tegument and envelope at a later stage of viral assembly.

Capsid↗

PubMed1987

Is Sindbis a simple picornavirus with an envelope?

A three-dimensional image reconstruction was performed from cryo-electron micrographs of isolated Sindbis (SNV) nucleocapsids. The isolated capsid is a smooth but fenestrated T = 3 structure. Comparison with the nucleocapsid seen within the whole virion indicated that the structure resembles the swollen forms which some non-enveloped viruses adopt after removal of divalent cations. A sensitive comparison method was used to align SNV capsid protein sequences with those of picornavirus vp3 capsid proteins whose high resolution structures display an eight-stranded beta-barrel fold found in many icosahedral viruses. The alignment predicted a similar folding for the Sindbis protein which juxtaposes several sets of residues known to be essential for its serine proteolytic activity. These results suggest that the capsid proteins of the enveloped alphaviruses and the non-enveloped picornaviruses may have arisen through divergent evolution from a simple, vp3-like ancestor.

Amino Acid Sequence↗

PubMed1997

Intermediates in the assembly pathway of the double-stranded RNA virus phi6.

The double-stranded RNA bacteriophage phi6 contains a nucleocapsid enclosed by a lipid envelope. The nucleocapsid has an outer layer of protein P8 and a core consisting of the four proteins P1, P2, P4 and P7. These four proteins form the polyhedral structure which acts as the RNA packaging and polymerase complex. Simultaneous expression of these four proteins in Escherichia coli gives rise to procapsids that can carry out the entire RNA replication cycle. Icosahedral image reconstruction from cryo-electron micrographs was used to determine the three-dimensional structures of the virion-isolated nucleocapsid and core, and of several procapsid-related particles expressed and assembled in E. coli. The nucleocapsid has a T = 13 surface lattice, composed primarily of P8. The core is a rounded structure with turrets projecting from the 5-fold vertices, while the procapsid is smaller than the core and more dodecahedral. The differences between the core and the procapsid suggest that maturation involves extensive structural rearrangements producing expansion. These rearrangements are co-ordinated with the packaging and RNA polymerization reactions that result in virus assembly. This structural characterization of the phi6 assembly intermediates reveals the ordered progression of obligate stages leading to virion assembly along with striking similarities to the corresponding Reoviridae structures.

Amino Acid Sequence↗

PubMed1992

Structural alterations of double-stranded DNA in complex with the adenovirus DNA-binding protein. Implications for its function in DNA replication.

The Adenovirus DNA-binding protein (DBP) binds to single-stranded (ss) DNA as well as to double-stranded (ds) DNA and forms multimeric protein-DNA complexes with both. Gel retardation assays indicate rapid complex formation for both DNAs. DBP rapidly dissociates from dsDNA, indicating a dynamic equilibrium, whereas the ssDNA-DBP complex is much more stable. We investigated the complex between DBP and dsDNA in more detail. Electron microscopical analysis shows thick filament-like and beaded structures in which the length of the DNA is not significantly altered. Cryo-electron micrographs suggest the presence of interwound protein fibres around the DNA. Ligase-mediated cyclization, but not linear multimerization, of DBP-saturated DNA fragments exceeding the persistence length was severely inhibited. This suggests that DNA may be organized by DBP into a rigid structure. Under those conditions, DBP induces distinct changes in the circular dichroism spectrum of the DNA, indicative of structural DNA changes. No bending or twisting of the complex was observed. Hydroxyl radical footprinting showed that the breakdown pattern of DNA at saturating DBP concentrations is much more regular than the protein-free DNA. This suggests the removal of tertiary structures, which may be related to the effects of DBP on enhanced NFI binding and chain elongation during Adenovirus DNA replication. Using purified proteins in an in vitro replication system, we correlate the structural changes with the effects of DBP on enhancement of NFI-binding as well as on DNA replication.

Adenoviruses, Human↗

PubMed1995

Proteolytic and conformational control of virus capsid maturation: the bacteriophage HK97 system.

Bacteriophage capsid assembly pathways provide excellent model systems to study large-scale conformational changes and other mechanisms that regulate the formation of macromolecular complexes. These capsids are formed from proheads: relatively fragile precursor particles which mature by undergoing extensive remodeling. Phage HK97 employs novel features in its strategy for building capsids, including assembly without a scaffolding protein, and the formation of a network of covalent cross-links between neighboring subunits in the mature virion. In addition, proteolytic cleavage of the capsid protein from 42 kDa to 31 kDa is essential for maturation. To investigate the structural bases for proteolysis and cross-linking, we have used cryo-electron micrographs to reconstruct the three-dimensional structures of purified particles from four discrete stages in the assembly pathway: Prohead I, Prohead II, Head I and Head II. Prohead I has icosahedral T = 7 packing of blister-shaped pentamers and hexamers. The pentamers are 5-fold symmetric, but the hexamers exhibit an unusual departure from 6-fold symmetry, as if two trimers had undergone a shear dislocation of about 25 A. Proteolytic conversion to Prohead II leaves the outer surface largely unchanged, but a major loss of density from the inner surface is observed, which we infer to represent the excision of the amino-terminal domains of the capsid protein. Upon expansion to the Head I state, the capsid becomes markedly larger, thinner walled, and more polyhedral: moreover, the capsomer shapes change radically; especially notable is the disappearance of the large hexon dislocation. No differences between Head I and the covalently cross-linked Head II could be observed at the current resolution of about 25 A, from which we infer that it is the conformational rearrangements effected by expansion that create the micro-environments needed for the autocatalytic formation of the isodipeptide bonds found in the mature virions ("pseudo-active sites").

Amino Acid Sequence↗

PubMed1996

The herpes simplex virus procapsid: structure, conformational changes upon maturation, and roles of the triplex proteins VP19c and VP23 in assembly.

The proteins coded by the five major capsid genes of herpes simplex virus 1, VP5 (gene UL19), VP19c (UL38), VP23 (UL18), pre-VP22a (UL26.5), and pre-VP21 (UL26), assemble into fragile roundish "procapsids", which mature into robust polyhedral capsids in a transition similar to that undergone by bacteriophage proheads. Here we describe the HSV-1 procapsid structure to a resolution of approximately 2.7 nm from three-dimensional reconstructions of cryo-electron micrographs. Comparison with the mature capsid provides insight into the large-scale conformational changes that take place upon maturation. In the procapsid, the elongated protomers (VP5 subunits) make little contact with each other except around the bases of the hexons and pentons, whereas they are tightly clustered into capsomers in the mature state; the axial channels, which are constricted or blocked in the mature capsid, are fully open; and unlike the well observed 6-fold symmetry of mature hexons, procapsid hexons are distorted into oval and triangular shapes. These deformations reveal a VP5 domain in the inner part of the protrusion wall which participates in inter-protomer bonding in the procapsid and is close to the site where the channel closes upon maturation. Remarkably, there are no direct contacts between neighboring capsomers; instead, interactions between them are mediated by the "triplexes" at the sites of local 3-fold symmetry. This observation discloses the mechanism whereby the triplex proteins, VP19c and VP23, play their essential roles in capsid morphogenesis. In the mature capsid, density extends continuously between neighboring capsomers in the inner "floor" layer. In contrast, there are large gaps in the corresponding region of the procapsid, implying that formation of the floor involves extensive remodeling. Inside the procapsid shell is the hollow spherical scaffold, whose radial density profile indicates that the major scaffold protein, pre-VP22a, is a long molecule (> 24 nm) composed of three domains. Since no evidence of icosahedral symmetry is detected in the scaffold, we infer that (unless higher resolution is required) the scaffold may not be an icosahedral shell but may instead be a protein micelle with a preferred radius of curvature.

Antibodies, Monoclonal↗

PubMedDate not supplied

Maximum-entropy three-dimensional reconstruction with deconvolution of the contrast transfer function: a test application with adenovirus.

We have developed an objective, quantitative, and general algorithm to improve the fidelity of three-dimensional reconstructions made from electron micrographs while at the same time filtering much of the noise present in the recorded data. The new technique is called constrained maximum entropy tomography (COMET). The essence of the method is that it will produce the most featureless reconstruction that fits the projection data within their observational accuracy. In particular, the COMET procedure will minimise the detrimental effects of errors in the measured data and deconvolute the effects of the contrast transfer function. An objective test has been performed using COMET on a conventional image reconstruction obtained from cryo-electron micrographs of adenovirus. The density for hexon, the major coat protein of the virus, which is known to high resolution from X-ray crystallography, provided a known high-resolution control. The COMET reconstruction is in considerably better agreement with the crystallographic electron density than the original reconstruction, throughout the entire resolution range.

Adenoviridae↗

PubMed1998

Three-dimensional structure of HIV-1 Rev protein filaments.

The HIV-1 Rev protein facilitates the export of incompletely spliced and unspliced viral mRNAs from the nucleus. Rev polymerizes into two types of filaments in vitro. In the presence of RNA, Rev forms poorly ordered structures, while in the absence of RNA it polymerizes into regular hollow filaments. We have determined the helical structure of the latter filaments by analysis of cryo-electron micrographs, taking into account STEM measurements of mass-per-unit-length. They are made up of Rev dimers, arranged in a six-start helix, with 31 dimers in 2 turns, a pitch angle of 45 degrees, and an interstrand spacing of 3.8 nm. Three-dimensional reconstruction at 2.1 nm resolution reveals a smooth outer surface and a featured inner surface, with outer and inner diameters of approximately 14.8 and approximately 10.4 nm, respectively. The Rev dimer has a "top-hat" shape with a cylinder approximately 3.2 nm in diameter and approximately 2.2 nm high, pointing inward: the thinner rim areas pack together to form the filament wall. Raman spectroscopy shows polymerized Rev to have approximately 54% alpha-helix and 20-24% beta-sheet content. Electron microdiffraction of aligned filaments reveals a broad meridional reflection at approximately (0.51 nm(-1, suggesting approximate alignment of the alpha-helices with the filament axis. Based on these data, a molecular model for the Rev filament is proposed.

Dimerization↗

PubMed1990

Cryo-electron microscopic studies of relaxed striated muscle thick filaments.

Electron micrograph images of rapidly frozen suspensions of thick filaments from four different muscle types are presented. Their optical and computer transforms are compared with images and diffraction patterns of negatively stained filaments and with X-ray data from the same muscles. We conclude that myosin head arrangement can be preserved on rapid freezing and that the images produced can be analysed by image processing techniques to give new information on thick filament structure.

Actin Cytoskeleton↗