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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↗

PubMed2000

Molecular architecture of bacteriophage T4 capsid: vertex structure and bimodal binding of the stabilizing accessory protein, Soc.

T4 encodes two dispensable proteins that bind to the outer surface of the mature capsid. Soc (9 kDa) stabilizes the capsid against extremes of alkaline pH and temperature, but Hoc (40 kDa) has no perceptible effect. Both proteins have been developed as display platforms. Their positions on the hexagonal surface lattice of gp23*, the major capsid protein, were previously defined by two-dimensional image averaging of negatively stained electron micrographs of elongated variant capsids. We have extended these observations by reconstructing cryo-electron micrographs of isometric capsids produced by a point mutant in gene 23, for both Hoc+.Soc+ and Hoc+.Soc- phages. The expected T = 13 lattice was observed, with a single Hoc molecule at the center of each gp23* hexamer. The vertices are occupied by pentamers of gp24*: despite limited sequence similarity with gp23*, the respective monomers are similar in size and shape, suggesting they may have the same fold. However, gp24* binds neither Hoc nor Soc; in situ, Soc is visualized as trimers at the trigonal points of the gp23* lattice and as monomers at the sites closest to the vertices. In solution, Soc is a folded protein ( approximately 10% alpha-helix and 50-60% beta sheet) that is monomeric as determined by analytic ultracentrifugation. Thus its trimerization on the capsid surface is imposed by a template of three symmetry-related binding sites. The observed mode of Soc binding suggests that it stabilizes the capsid by a clamping mechanism and offers a possible explanation for the phenotype of osmotic shock resistance.

Bacteriophage T4↗

PubMed1998

Limited flexibility of the inter-protofilament bonds in microtubules assembled from pure tubulin.

The superposition of the regular arrangement of tubulin subunits in microtubules gives rise to moiré patterns in cryo-electron micrographs. The moiré period can be predicted from the dimensions of the tubulin subunits and their arrangement in the surface lattice. Although the average experimental moiré period is usually in good agreement with the theoretical one, there is variation both within and between microtubules. In this study, we addressed the origin of this variability. We examined different possibilities, including artefacts induced by the preparation of the vitrified samples, and variations of the parameters that describe the microtubule surface lattice. We show that neither flattening nor bending of the microtubules, nor changes in the subunit dimensions, can account for the moiré period variations observed in 12 and 14 protofilament microtubules. These can be interpreted as slight variations, in the range -0.5 A to +0.9 A, of the lateral interactions between tubulin subunits in adjacent protofilaments. These results indicate that the inter-protofilament bonds are precisely maintained in microtubules assembled in vitro from pure tubulin. The fact that the moiré period is not affected by bending indicates that the local interactions between tubulin subunits are sufficiently stiff to accommodate large deformations of the microtubule wall.

Animals↗

PubMed1999

A symmetry mismatch at the site of RNA packaging in the polymerase complex of dsRNA bacteriophage phi6.

The polymerase complex of the enveloped double-stranded RNA (dsRNA) bacteriophage phi6 fulfils a similar function to those of other dsRNA viruses such as Reoviridae. The phi6 complex comprises protein P1, which forms the shell, and proteins P2, P4 and P7, which are involved in RNA synthesis and packaging. Icosahedral reconstructions from cryo-electron micrographs of recombinant polymerase particles revealed a clear dodecahedral shell and weaker satellites. Difference imaging demonstrated that these weak satellites were the sites of P4 and P2 within the complex. The structure determined by icosahedral reconstruction was used as an initial model in an iterative reconstruction technique to examine the departures from icosahedral symmetry. This approach showed that P4 and P2 contribute to structures at the 5-fold positions of the icosahedral P1 shell which lack 5-fold symmetry and appear in variable orientations. Reconstruction of isolated recombinant P4 showed that it was a hexamer with a size and shape matching the satellite. Symmetry mismatch between the satellites and the shell could play a role in RNA packaging akin to that of the portal vertex of dsDNA phages in DNA packaging. This is the first example of dsRNA virus in which the structure of the polymerase complex has been determined without the assumption of icosahedral symmetry. Our result with phi6 illustrates the symmetry mismatch which may occur at the sites of RNA packaging in other dsRNA viruses such as members of the Reoviridae.

Bacteriophage phi 6↗

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↗

PubMed2000

Digitally collected cryo-electron micrographs for single particle reconstruction.

Several advantages and disadvantages have been cited for image collection with a slow-scan CCD camera. Here we explore its use for cryo-EM single particle reconstruction and present two practical examples. The icosahedral adenovirus (Ad) type 2 ( approximately 150 MDa) was reconstructed from 396 particle images. The Fourier shell correlation (FSC) 0.5 threshold and the Fourier shell phase residual (FSPR) 45 degrees criterion yielded 17 AA resolution for the ordered viral capsid. Visual comparison with the filtered Ad2 crystallographic hexon confirmed a resolution range of 15-17 A. The asymmetric DNA-PKcs protein (470 kDa) was reconstructed from 9,473 particle images, using a previously published reconstruction based on class-sum images as an orientational search model [Chiu et al. (1998) J. Mol. Biol. 284:1075-1081]. FSC and FSPR methods yielded 17 A resolution for the new DNA-PKcs reconstruction, indicating a small but noticeable improvement over that of the class-sum based reconstruction. Despite the lack of symmetry for DNA-PKcs and its lower image contrast compared to Ad2 (0.8% vs. 2.5%), the same resolution was obtained for both particles by averaging significantly more DNA-PKcs images. Use of the CCD camera enables the microscopist to adjust the electron beam strength interactively and thereby maximize the image contrast for beam sensitive samples. On-line Fourier transformation also allows routine monitoring of drift and astigmatism during image collection, resulting in a high percentage of micrographs suitable for image processing. In conclusion, our results show that digital image collection with the YAG-scintillator slow-scan CCD camera is a viable approach for 3D reconstruction of both symmetric and asymmetric particles.

Adenoviruses, Human↗

PubMed2000

The 3D arrangement of the 23 S and 5 S rRNA in the Escherichia coli 50 S ribosomal subunit based on a cryo-electron microscopic reconstruction at 7.5 A resolution.

The Escherichia coli 23 S and 5 S rRNA molecules have been fitted helix by helix to a cryo-electron microscopic (EM) reconstruction of the 50 S ribosomal subunit, using an unfiltered version of the recently published 50 S reconstruction at 7.5 A resolution. At this resolution, the EM density shows a well-defined network of fine structural elements, in which the major and minor grooves of the rRNA helices can be discerned at many locations. The 3D folding of the rRNA molecules within this EM density is constrained by their well-established secondary structures, and further constraints are provided by intra and inter-rRNA crosslinking data, as well as by tertiary interactions and pseudoknots. RNA-protein cross-link and foot-print sites on the 23 S and 5 S rRNA were used to position the rRNA elements concerned in relation to the known arrangement of the ribosomal proteins as determined by immuno-electron microscopy. The published X-ray or NMR structures of seven 50 S ribosomal proteins or RNA-protein complexes were incorporated into the EM density. The 3D locations of cross-link and foot-print sites to the 23 S rRNA from tRNA bound to the ribosomal A, P or E sites were correlated with the positions of the tRNA molecules directly observed in earlier reconstructions of the 70 S ribosome at 13 A or 20 A. Similarly, the positions of cross-link sites within the peptidyl transferase ring of the 23 S rRNA from the aminoacyl residue of tRNA were correlated with the locations of the CCA ends of the A and P site tRNA. Sites on the 23 S rRNA that are cross-linked to the N termini of peptides of different lengths were all found to lie within or close to the internal tunnel connecting the peptidyl transferase region with the presumed peptide exit site on the solvent side of the 50 S subunit. The post-transcriptionally modified bases in the 23 S rRNA form a cluster close to the peptidyl transferase area. The minimum conserved core elements of the secondary structure of the 23 S rRNA form a compact block within the 3D structure and, conversely, the points corresponding to the locations of expansion segments in 28 S rRNA all lie on the outside of the structure.

Base Sequence↗

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↗

PubMed1999

Methods for reconstructing density maps of "single" particles from cryoelectron micrographs to subnanometer resolution.

Recently the resolution attainable in density maps calculated from cryo-electron micrographs of free-standing virus capsids has advanced to resolutions below 1 nm. This represents a significant milestone in that resolutions of this order potentially allow direct visualization of individual elements of protein secondary structure (i.e., alpha-helices), in addition to the shapes and connectivity of subdomains. We describe here a computational strategy for structural analyses at this level of detail: its principal innovation is a procedure for correcting the contrast transfer function of the electron microscope. Also important is the practice of combining data from pairs of differently defocused micrographs to improve the signal-to-noise ratio of the images, thereby allowing more precise determinations of the particles' orientations and origins and contributing to higher resolution reconstructions. These procedures proved instrumental in our analysis of the capsid of hepatitis B virus at 9-A resolution (Conway et al., 1997, Nature 386, 91-94). Finally, we discuss the prospects for achieving comparable resolutions for isolated macromolecular complexes with lower symmetry or no symmetry and for further extension of the resolution.

Capsid↗

PubMed2000

The cell surface glycoprotein layer of the extreme halophile Halobacterium salinarum and its relation to Haloferax volcanii: cryo-electron tomography of freeze-substituted cells and projection studies of negatively stained envelopes.

We have studied the surface layer (S-layer) of Halobacterium salinarum (formerly Halobacterium halobium), an extreme halophile requiring high concentrations of sodium, by electron microscopy of (a) isolated, negatively stained, flattened envelopes and (b) cryo-fixation of intact cells in their high-salt growth medium followed by freeze substitution and tomography of thin sections. From the negatively stained isolated envelopes we have calculated a two-dimensional, projection map that is strikingly similar to that of Haloferax volcanii, an extreme halophile requiring high concentrations of magnesium; both projection maps show the hexagonal arrangement of the morphological units with an identical center-to-center spacing of 150 A; each of the morphological units of the two species has six subunits with a similar density distribution and apparent domain organization. In contrast to the two-dimensional map, the tomographic reconstruction of Halob. salinarum does not agree in a straightforward way with the three-dimensional, electron crystallographic map of negatively stained Halof. volcanii envelopes, although the main features of the lattice and the morphological units are evident. The tomographic reconstruction of sections from epoxy-embedded material suffers from directional compression due to sectioning stress and continuous dimensional changes and mass loss due to electron irradiation. This communication consists, therefore, of three parts: (a) a comparison of the projection maps of negatively stained envelopes of Halof. volcanii and Halob. salinarum; (b) a comparison of the three-dimensional maps obtained by electron crystallography (Halof. volcanii) and low-dose cryo-tomography (Halob. salinarum); and (c) a methodological study of mass loss and dimensional changes of plastic-embedded material under low-dose conditions at room and liquid nitrogen temperatures.

Archaeal Proteins↗

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↗

PubMed1995

Cryo-electron energy loss spectroscopy: observations on vitrified hydrated specimens and radiation damage.

Valence electron energy loss spectroscopy (EELS) has been used to characterize the composition of frozen-hydrated specimens in the electron microscope. Fine structure in the energy range up to 30 eV provides a means of distinguishing between vitreous and crystalline ice. Some features of the ice spectrum can be understood in terms of transitions between molecular orbitals in the water molecule and by the existence of excitons in the solid. Spectra from hydrated biological specimens can be analyzed to obtain quantitative estimates of the water content by fitting contributions from the ice and organic components. EELS also provides information about the radiation chemistry that occurs when hydrated specimens are exposed to the electron beam. From the observation of the hydrogen K-edge at approximately 13 eV, it can be deduced that bubbles of molecular hydrogen are evolved during irradiation at doses of > 10(4) nm-2, and that these bubbles contain gas at pressures in excess of one thousand atmospheres.

Animals↗