Spectral signal-to-noise ratio and resolution assessment of 3D reconstructions.

Measuring the quality of three-dimensional (3D) reconstructed biological macromolecules by transmission electron microscopy is still an open problem. In this article, we extend the applicability of the spectral signal-to-noise ratio (SSNR) to the evaluation of 3D volumes reconstructed with any reconstruction algorithm. The basis of the method is to measure the consistency between the data and a corresponding set of reprojections computed for the reconstructed 3D map. The idiosyncrasies of the reconstruction algorithm are taken explicitly into account by performing a noise-only reconstruction. This results in the definition of a 3D SSNR which provides an objective indicator of the quality of the 3D reconstruction. Furthermore, the information to build the SSNR can be used to produce a volumetric SSNR (VSSNR). Our method overcomes the need to divide the data set in two. It also provides a direct measure of the performance of the reconstruction algorithm itself; this latter information is typically not available with the standard resolution methods which are primarily focused on reproducibility alone.

Capsid structure of Kaposi's sarcoma-associated herpesvirus, a gammaherpesvirus, compared to those of an alphaherpesvirus, herpes simplex virus type 1, and a betaherpesvirus, cytomegalovirus.

The capsid of Kaposi's sarcoma-associated herpesvirus (KSHV) was visualized at 24-A resolution by cryoelectron microscopy. Despite limited sequence similarity between corresponding capsid proteins, KSHV has the same T=16 triangulation number and much the same capsid architecture as herpes simplex virus (HSV) and cytomegalovirus (CMV). Its capsomers are hexamers and pentamers of the major capsid protein, forming a shell with a flat, close-packed, inner surface (the "floor") and chimney-like external protrusions. Overlying the floor at trigonal positions are (alpha beta(2)) heterotrimers called triplexes. The floor structure is well conserved over all three viruses, and the most variable capsid features reside on the outer surface, i.e., in the shapes of the protrusions and triplexes, in which KSHV resembles CMV and differs from HSV. Major capsid protein sequences from the three subfamilies have some similarity, which is closer between KSHV and CMV than between either virus and HSV. The triplex proteins are less highly conserved, but sequence analysis identifies relatively conserved tracts. In alphaherpesviruses, the alpha-subunit (VP19c in HSV) has a 100-residue N-terminal extension and an insertion near the C terminus. The small basic capsid protein sequences are highly divergent: whereas the HSV and CMV proteins bind only to hexons, difference mapping suggests that the KSHV protein, ORF65, binds around the tips of both hexons and pentons.

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.

Three-dimensional structure of poliovirus receptor bound to poliovirus.

Poliovirus initiates infection by binding to its cellular receptor (Pvr). We have studied this interaction by using cryoelectron microscopy to determine the structure, at 21-A resolution, of poliovirus complexed with a soluble form of its receptor (sPvr). This density map aided construction of a homology-based model of sPvr and, in conjunction with the known crystal structure of the virus, allowed delineation of the binding site. The virion does not change significantly in structure on binding sPvr in short incubations at 4 degrees C. We infer that the binding configuration visualized represents the initial interaction that is followed by structural changes in the virion as infection proceeds. sPvr is segmented into three well-defined Ig-like domains. The two domains closest to the virion (domains 1 and 2) are aligned and rigidly connected, whereas domain 3 diverges at an angle of approximately 60 degrees. Two nodules of density on domain 2 are identified as glycosylation sites. Domain 1 penetrates the "canyon" that surrounds the 5-fold protrusion on the capsid surface, and its binding site involves all three major capsid proteins. The inferred pattern of virus-sPvr interactions accounts for most mutations that affect the binding of Pvr to poliovirus.

Molecular tectonic model of virus structural transitions: the putative cell entry states of poliovirus.

Upon interacting with its receptor, poliovirus undergoes conformational changes that are implicated in cell entry, including the externalization of the viral protein VP4 and the N terminus of VP1. We have determined the structures of native virions and of two putative cell entry intermediates, the 135S and 80S particles, at approximately 22-A resolution by cryo-electron microscopy. The 135S and 80S particles are both approximately 4% larger than the virion. Pseudoatomic models were constructed by adjusting the beta-barrel domains of the three capsid proteins VP1, VP2, and VP3 from their known positions in the virion to fit the 135S and 80S reconstructions. Domain movements of up to 9 A were detected, analogous to the shifting of tectonic plates. These movements create gaps between adjacent subunits. The gaps at the sites where VP1, VP2, and VP3 subunits meet are plausible candidates for the emergence of VP4 and the N terminus of VP1. The implications of these observations are discussed for models in which the externalized components form a transmembrane pore through which viral RNA enters the infected cell.

Isolation of herpes simplex virus procapsids from cells infected with a protease-deficient mutant virus.

Herpes simplex virus type 1 (HSV-1) capsid proteins assemble in vitro into spherical procapsids that differ markedly in structure and stability from mature polyhedral capsids but can be converted to the mature form. Circumstantial evidence suggests that assembly in vivo follows a similar pathway of procapsid assembly and maturation, a pathway that resembles those of double-stranded DNA bacteriophages. We have confirmed the above pathway by isolating procapsids from HSV-1-infected cells and characterizing their morphology, thermal sensitivity, and protein composition. Experiments were carried out with an HSV-1 mutant (m100) deficient in the maturational protease for which it was expected that procapsids-normally, short-lived intermediates-would accumulate in infected cells. Particles isolated from m100-infected cells were found to share the defining properties of procapsids assembled in vitro. For example, by electron microscopy, they were found to be spherical rather than polyhedral in shape, and they disassembled at 0 degrees C, unlike mature capsids, which are stable at this temperature. A three-dimensional reconstruction computed at 18-A resolution from cryoelectron micrographs showed m100 procapsids to be structurally indistinguishable from procapsids assembled in vitro. In both cases, their predominant components are the four essential capsid proteins: the major capsid protein (VP5), the scaffolding protein (pre-VP22a), and the triplex proteins (VP19C and VP23). VP26, a small, abundant but dispensable capsid protein, was not found associated with m100 procapsids, suggesting that it binds to capsids only after they have matured into the polyhedral form. Procapsids were also isolated from cells infected at the nonpermissive temperature with the HSV-1 mutant tsProt.A (a mutant with a thermoreversible lesion in the protease), and their identity as procapsids was confirmed by cryoelectron microscopy. This analysis revealed density on the inner surface of the procapsid scaffolding core that may correspond to the location of the maturational protease. Upon incubation at the permissive temperature, tsProt.A procapsids transformed into polyhedral, mature capsids, providing further confirmation of their status as precursors.

A strategy for determining the orientations of refractory particles for reconstruction from cryo-electron micrographs with particular reference to round, smooth-surfaced, icosahedral viruses.

Cryo-electron microscopy and three-dimensional image reconstruction are powerful tools for analyzing icosahedral virus capsids at resolutions that now extend below 1 nm. However, the validity of such density maps depends critically on correct identification of the viewing geometry of each particle in the data set. In some cases-for example, round capsids with low surface relief-it is difficult to identify orientations by conventional application of the two most widely used approaches-"common lines" and model-based iterative refinement. We describe here a strategy for determining the orientations of such refractory specimens. The key step is to determine reliable orientations for a base set of particles. For each particle, a list of candidate orientations is generated by common lines: correct orientations are then identified by computing a single-particle reconstruction for each candidate and then systematically matching their reprojections with the original images by visual criteria and cross-correlation analysis. This base set yields a first-generation reconstruction that is fed into the model-based procedure. This strategy has led to the structural determination of two viruses that, in our hands, resisted solution by other means.

Assembly of the herpes simplex virus procapsid from purified components and identification of small complexes containing the major capsid and scaffolding proteins.

An in vitro system is described for the assembly of herpes simplex virus type 1 (HSV-1) procapsids beginning with three purified components, the major capsid protein (VP5), the triplexes (VP19C plus VP23), and a hybrid scaffolding protein. Each component was purified from insect cells expressing the relevant protein(s) from an appropriate recombinant baculovirus vector. Procapsids formed when the three purified components were mixed and incubated for 1 h at 37 degrees C. Procapsids assembled in this way were found to be similar in morphology and in protein composition to procapsids formed in vitro from cell extracts containing HSV-1 proteins. When scaffolding and triplex proteins were present in excess in the purified system, greater than 80% of the major capsid protein was incorporated into procapsids. Sucrose density gradient ultracentrifugation studies were carried out to examine the oligomeric state of the purified assembly components. These analyses showed that (i) VP5 migrated as a monomer at all of the protein concentrations tested (0.1 to 1 mg/ml), (ii) VP19C and VP23 migrated together as a complex with the same heterotrimeric composition (VP19C1-VP232) as virus triplexes, and (iii) the scaffolding protein migrated as a heterogeneous mixture of oligomers (in the range of monomers to approximately 30-mers) whose composition was strongly influenced by protein concentration. Similar sucrose gradient analyses performed with mixtures of VP5 and the scaffolding protein demonstrated the presence of complexes of the two having molecular weights in the range of 200,000 to 600,000. The complexes were interpreted to contain one or two VP5 molecules and up to six scaffolding protein molecules. The results suggest that procapsid assembly may proceed by addition of the latter complexes to regions of growing procapsid shell. They indicate further that procapsids can be formed in vitro from virus-encoded proteins only without any requirement for cell proteins.

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.

Two antibodies that neutralize papillomavirus by different mechanisms show distinct binding patterns at 13 A resolution.

Complexes between bovine papillomavirus type 1 (BPV1) and examples of two sets of neutralizing, monoclonal antibodies (mAb) to the major capsid protein (L1) were analyzed by low-dose cryo-electron microscopy and three-dimensional (3D) image reconstruction to 13 A resolution. mAb #9 is representative of a set of neutralizing antibodies that can inhibit viral binding to the cell surface, while mAb 5B6 is representative of a second set that efficiently neutralizes papillomaviruses without significantly inhibiting viral binding to the cell surface. The 3D reconstructions reveal that mAb #9 binds to L1 molecules of both pentavalent and hexavalent capsomeres. In contrast, 5B6 binds only to hexavalent capsomeres, reflecting the significant structural or environmental differences for the 5B6 epitope in the 12 pentavalent capsomeres. Epitope localization shows that mAb #9 binds monovalently to the tips of capsomeres whereas 5B6 binds both monovalently and bivalently to the sides of hexavalent capsomeres approximately two-thirds of the way down from the outer tips, very close to the putative stabilizing intercapsomere connections. The absence of mAb 5B6 from the pentavalent capsomeres and its inability to prevent viral binding to the cell surface suggest that receptor binding may occur at one or more of the 12 virion vertices.

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.

Hexon-only binding of VP26 reflects differences between the hexon and penton conformations of VP5, the major capsid protein of herpes simplex virus.

VP26 is a 12-kDa capsid protein of herpes simplex virus 1. Although VP26 is dispensable for assembly, the native capsid (a T=16 icosahedron) contains 900 copies: six on each of the 150 hexons of VP5 (149 kDa) but none on the 12 VP5 pentons at its vertices. We have investigated this interaction by expressing VP26 in Escherichia coli and studying the properties of the purified protein in solution and its binding to capsids. Circular dichroism spectroscopy reveals that the conformation of purified VP26 consists mainly of beta-sheets (approximately 80%), with a small alpha-helical component (approximately 15%). Its state of association was determined by analytical ultracentrifugation to be a reversible monomer-dimer equilibrium, with a dissociation constant of approximately 2 x 10(-5) M. Bacterially expressed VP26 binds to capsids in the normal amount, as determined by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cryoelectron microscopy shows that the protein occupies its usual sites on hexons but does not bind to pentons, even when available in 100-fold molar excess. Quasi-equivalence requires that penton VP5 must differ in conformation from hexon VP5: our data show that in mature capsids, this difference is sufficiently pronounced to abrogate its ability to bind VP26.

Structure of L-A virus: a specialized compartment for the transcription and replication of double-stranded RNA.

The genomes of double-stranded (ds)RNA viruses are never exposed to the cytoplasm but are confined to and replicated from a specialized protein-bound compartment-the viral capsid. We have used cryoelectron microscopy and three-dimensional image reconstruction to study this compartment in the case of L-A, a yeast virus whose capsid consists of 60 asymmetric dimers of Gag protein (76 kD). At 16-A resolution, we distinguish multiple domains in the elongated Gag subunits, whose nonequivalent packing is reflected in subtly different morphologies of the two protomers. Small holes, 10-15 A across, perforate the capsid wall, which functions as a molecular sieve, allowing the exit of transcripts and the influx of metabolites, while retaining dsRNA and excluding degradative enzymes. Scanning transmission electron microscope measurements of mass-per-unit length suggest that L-A RNA is an A-form duplex, and that RNA filaments emanating from disrupted virions often consist of two or more closely associated duplexes. Nuclease protection experiments confirm that the genome is entirely sequestered inside full capsids, but it is packed relatively loosely; in L-A, the center-to-center spacing between duplexes is 40-45 A, compared with 25-30 A in other double-stranded viruses. The looser packing of L-A RNA allows for maneuverability in the crowded capsid interior, in which the genome (in both replication and transcription) must be translocated sequentially past the polymerase immobilized on the inner capsid wall.

The making and breaking of symmetry in virus capsid assembly: glimpses of capsid biology from cryoelectron microscopy.

Virus capsids constitute a diverse and versatile family of protein-bound containers and compartments ranging in diameter from approximately 200 A (mass approximately 1 MDa) to >1500 A (mass>250 MDa). Cryoelectron microscopy of capsids, now attaining resolutions down to 10 A, is disclosing novel structural motifs, assembly mechanisms, and the precise locations of major epitopes. Capsids are essentially symmetric structures, and icosahedral surface lattices have proved to be widespread. However, many capsid proteins exhibit a remarkable propensity for symmetry breaking, whereby chemically identical subunits in distinct lattice sites have markedly different structures and packing relationships. Temporal differences in the conformation of a given subunit are also manifested in the large-scale conformational changes that accompany capsid maturation. Larger and more complex capsids, such as DNA bacteriophages and herpes simplex virus, are formed not by simple self-assembly, but under the control of tightly regulated programs that may include the involvement of viral scaffolding proteins and cellular chaperonins, maturational proteolysis, and conformational changes on an epic scale. In addition to its significance for virology, capsid-related research has implications for biology in general, relating to the still largely obscure assembly processes of macromolecular complexes that perform many important cellular functions.

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.

Structure of the herpes simplex virus capsid: peptide A862-H880 of the major capsid protein is displayed on the rim of the capsomer protrusions.

The herpes simplex virus-1 (HSV-1) capsid shell has 162 capsomers arranged on a T = 16 icosahedral lattice. The major capsid protein, VP5 MW = 149,075) is the structural component of the capsomers. VP5 is an unusually large viral capsid protein and has been shown to consist of multiple domains. To study the conformation of VP5 as it is folded into capsid promoters, we identified the sequence recognized by a VP5-specific monoclonal antibody and localized the epitope on the capsid surface by cryoelectron microscopy and image reconstruction. The epitope of mAb 6F10 was mapped to residues 862-880 by immunoblotting experiments performed with (1) proteolytic fragments of VP5, (2) GST-fusion proteins containing VP5 domains, and (3) synthetic VP5 peptides. As visualized in a three-dimensional density map of 6F10-precipitated capsids, the antibody was found to bind at sites on the outer surface of the capsid just inside the openings of the trans-capsomeric channels. We conclude that these sites are occupied by peptide 862-880 in the mature HSV-1 capsid.

Assembly of the herpes simplex virus capsid: characterization of intermediates observed during cell-free capsid formation.

The herpes simplex virus-1 (HSV-1) capsid is an icosahedral shell approximately 15 nm thick and 125 nm in diameter. Three of its primary structural components are a major capsid protein (VP5; coded by the UL19 gene) and two minor proteins, VP19C (UL38 gene) and VP23 (UL18 gene). Assembly of the capsid involves the participation of two additional proteins, the scaffolding protein (UL26.5 gene) and the maturational protease (UL26 gene). With the goal of identifying morphological intermediates in the assembly process, we have examined capsid formation in a cell-free system containing the five HSV-1 proteins mentioned above. Capsids and capsid-related structures formed during progressively longer periods of incubation were examined by electron microscopy of thin-sectioned specimens. After one minute, 90 minutes and eight hours of incubation the structures observed, respectively, were partial capsids, closed spherical capsids and polyhedral capsids. Partial capsids were two-layered structures consisting of a segment of external shell partially surrounding a region of scaffold. They appeared as wedges or angular segments of closed spherical capsids, the angle ranging from less than 30 degrees to greater than 270 degrees. Partial capsids are suggested to be precursors of closed spherical capsids because, whereas partial capsids were the predominant assembly product observed after one minute of incubation, they were rare in reactions incubated for 45 minutes or longer. Closed spherical capsids were highly uniform in morphology, consisting of a closed external shell surrounding a thick scaffold similar in morphology to the same layers seen in partial capsids. In negatively stained specimens, closed spherical capsids appeared round in profile, suggesting that they are spherical rather than polyhedral in shape. A three-dimensional reconstruction computed from cryoelectron micrographs confirmed that closed spherical capsids are spherical with T = 16 icosahedral symmetry. The reconstruction showed further that, compared to mature HSV-1 capsids, closed spherical capsids are more open structures in which the capsid floor layer is less pronounced. In contrast to closed spherical capsids, polyhedral capsids exhibited distinct facets and vertices, indicating that they are icosahedral like the capsids in mature virions. Upon incubation in vitro, purified closed spherical capsids matured into polyhedral capsids, indicating that the latter arise by angularization of the former. Partial capsids, closed spherical capsids and polyhedral capsids were all found to contain VP5, VP19C, VP23, VP21 and the scaffolding protein; the scaffolding protein being predominantly in the immature, uncleaved form in all cases. Polyhedral capsids and closed spherical capsids were found to differ in their sensitivity to disruption at 2 degrees C. Closed spherical capsids were disassembled while polyhedral capsids were unaffected. Our results suggest that HSV-1 capsid assembly begins with the partial capsid and proceeds through a closed, spherical, unstable capsid intermediate to a closed, icosahedral form similar to that found in the mature virion. Structures resembling HSV-1 partial capsids have been described as capsid assembly intermediates in Salmonella typhimurium bacteriophage P22. HSV-1 capsid maturation from a fragile, spherical state to a robust polyhedral form resembles the prohead maturation events undergone by dsDNA bacteriophages including lambda, T4 and P22. Because of this similarity, we propose the name procapsid for the closed spherical capsid intermediate in HSV-1 capsid assembly.

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.

Constructing retinal fundus photomontages. A new computer-based method.

PURPOSE: To develop computer algorithms for reconstructing 24-bit color, wide-angle composite retinal fundus images from a set of adjacent 45 degrees fundus slides. The authors present the description, technical details, and results of the image reconstruction technique. METHODS: Patients with retinal degeneration underwent fundus photography with a 45 degrees field-of-view fundus camera. Individual photographic slides were digitized for creating fundus montages. Background variations in individual 45 degrees images were modeled to first- or second-order two-dimensional polynomial functions to generate a background image. The background image was subtracted from the original image to obtain background corrected image. Background corrected images were registered and spatially transformed using a first- or second-order two-dimensional polynomial warp model to reconstruct a composite retinal fundus montage. RESULTS: The authors successfully reconstructed 24-bit color, 100 degrees field-of-view, composite retinal fundus images. The computer-reconstructed montages are an improvement over manually generated montages because computer analysis can be performed on the computer-based montages. In addition, background variations and discontinuities between individual photographs observed in manually generated montages are reduced greatly in computer-generated montages. Most important, the computer-generated montages are better aligned than the manually generated photomontages. CONCLUSIONS: This method of reconstructing a wide-angle composite retinal fundus image from a set of adjacent small- and wide-angle fundus slides is a new tool for creating montages as large as 100 degrees field of view. The computer-generated montages may be used for documenting and quantifying retinal findings. This can greatly assist studies of retinal manifestations of diseases, such as gyrate atrophy, retinitis pigmentosa, sickle cell disease, and acquired immune deficiency syndrome.

Three-dimensional structure of Bordetella pertussis fimbriae.

We describe the helical structure of Bordetella pertussis fimbriae of serotype 3/6 as determined to a resolution of approximately 2.5 nm by three-dimensional reconstruction of negatively stained electron micrographs. The fimbria has a distinctly polar structure whose axial repeat of 13 nm contains five copies of the fim3 gene product (22 kDa) in two complete turns. These subunits are connected by interactions along the fimbrial backbone which, unlike other classes of bacterial fimbriae, has no axial channel. Its outer diameter is approximately 5.7 nm, and the most pronounced feature is a radially protruding domain that gives the fimbria its characteristic serrated appearance. Serotype 2 fimbriae, composed of the fim2 subunit which is 60% homologous with fim3, have essentially the same quaternary structure. These observations are discussed in relation to fimbrial phase variation and structure-based classification of fimbriae/pili.