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Stephen D Fuller

Publications and source records attributed to Stephen D Fuller.

18 recordsLinked to original sources
PubMed2006

Structure of a hexameric RNA packaging motor in a viral polymerase complex.

Packaging of the Cystovirus varphi8 genome into the polymerase complex is catalysed by the hexameric P4 packaging motor. The motor is located at the fivefold vertices of the icosahedrally symmetric polymerase complex, and the symmetry mismatch between them may be critical for function. We have developed a novel image-processing approach for the analysis of symmetry-mismatched structures and applied it to cryo-electron microscopy images of P4 bound to the polymerase complex. This approach allowed us to solve the three-dimensional structure of the P4 in situ to 15-A resolution. The C-terminal face of P4 was observed to interact with the polymerase complex, supporting the current view on RNA translocation. We suggest that the symmetry mismatch between the two components may facilitate the ring opening required for RNA loading prior to its translocation.

Cryoelectron Microscopy↗
PubMed2006

Determination of astigmatism in TEM images.

We have developed a new two-step algorithm to determine the astigmatism of images from transmission electron microscopes (TEMs). Instead of computing the radial average of the power spectrum, we divide the power spectrum of a TEM image 1 to m (typically 32) sectors. We use a technique based on perturbation analysis of the contrast transfer function (CTF) to assimilate sector averages of the power spectrum of an image, which are incoherent in the presence of astigmatism, to a coherent radial average corresponding to a nominal defocus value. This is based on the fact that small defocus change from a nominal value can be considered to be equivalent to a perturbation on the spatial frequency spectra. Thus, instead of measuring the angular defocus variations, we optimise the frequency change required to obtain a coherent radial average. Numerically, this is achieved by minimizing sigma(2)/sigma(1) of a matrix formed from the sector averages, where sigma(i) denotes the ith singular value of the matrix. After the minimisation procedure, the second singular value should be very small compared with the first singular value, indicating that the matrix is nearly rank unity. In the second step, the nominal defocus can be obtained from the coherent radial average using any good defocus estimation program, which assumes zero astigmatism. The defocus value at a sector can be obtained from this nominal defocus value and one of the parameters from the unconstrained optima. Our algorithm is tested on astigmatic images of carbon film, 2D crystals of bacteriorhodopsin and cryo-images of HIV cores.

Algorithms↗
PubMed2006

The mechanism of HIV-1 core assembly: insights from three-dimensional reconstructions of authentic virions.

Infectious HIV particles contain a characteristic cone-shaped core encasing the viral RNA and replication proteins. The core exhibits significant heterogeneity in size and shape, yet consistently forms a well-defined structure. The mechanism by which the core is assembled in the maturing virion remains poorly understood. Using cryo-electron tomography, we have produced three-dimensional reconstructions of authentic, unstained HIV-1. These reveal the viral morphology with unprecedented clarity and suggest the following mechanism for core formation inside the extracellular virion: core growth initiates at the narrow end of the cone and proceeds toward the distal side of the virion until limited by the viral membrane. Curvature and closure of the broad end of the core are then directed by the inner surface of the viral membrane. This mechanism accommodates significant flexibility in lattice growth while ensuring the closure of cores of variable size and shape.

Cryoelectron Microscopy↗
PubMed2006

Structure of the bacteriophage phi6 nucleocapsid suggests a mechanism for sequential RNA packaging.

Bacteriophage phi6 is an enveloped dsRNA virus with a segmented genome. Phi6 specifically packages one copy of each of its three genome segments into a preassembled polymerase complex. This leads to expansion of the polymerase complex, minus and plus strand RNA synthesis, and assembly of the nucleocapsid. The phi6 in vitro assembly and packaging system is a valuable model for dsRNA virus replication. The structure of the nucleocapsid at 7.5 A resolution presented here reveals the secondary structure of the two major capsid proteins. Asymmetric P1 dimers organize as an inner T = 1 shell, and P8 trimers organize as an outer T = 13 laevo shell. The organization of the P1 molecules in the unexpanded and expanded polymerase complex suggests that the expansion is accomplished by rigid body movements of the P1 monomers. This leads to exposure of new potential RNA binding surfaces to control the sequential packaging of the genome segments.

Bacteriophage phi 6↗
PubMed2006

Cryo-electron tomographic structure of an immunodeficiency virus envelope complex in situ.

The envelope glycoprotein (Env) complexes of the human and simian immunodeficiency viruses (HIV and SIV, respectively) mediate viral entry and are a target for neutralizing antibodies. The receptor binding surfaces of Env are in large part sterically occluded or conformationally masked prior to receptor binding. Knowledge of the unliganded, trimeric Env structure is key for an understanding of viral entry and immune escape, and for the design of vaccines to elicit neutralizing antibodies. We have used cryo-electron tomography and averaging to obtain the structure of the SIV Env complex prior to fusion. Our result reveals novel details of Env organisation, including tight interaction between monomers in the gp41 trimer, associated with a three-lobed, membrane-distal gp120 trimer. A cavity exists at the gp41-gp120 trimer interface. Our model for the spike structure agrees with previously predicted interactions between gp41 monomers, and furthers our understanding of gp120 interactions within an intact spike.

Animals↗
PubMed2005

Cryo-electron microscopy reveals conserved and divergent features of gag packing in immature particles of Rous sarcoma virus and human immunodeficiency virus.

Retrovirus assembly proceeds via multimerisation of the major structural protein, Gag, into a tightly packed, spherical particle that buds from the membrane of the host cell. The lateral packing arrangement of the human immunodeficiency virus type 1 (HIV-1) Gag CA (capsid) domain in the immature virus has been described. Here we have used cryo-electron microscopy (cryo-EM) and image processing to determine the lateral and radial arrangement of Gag in in vivo and in vitro assembled Rous sarcoma virus (RSV) particles and to compare these features with those of HIV-1. We found that the lateral packing arrangement in the vicinity of the inner sub-domain of CA is conserved between these retroviruses. The curvature of the lattice, however, is different. RSV Gag protein adopts a more tightly curved lattice than is seen in HIV-1, and the virions therefore contain fewer copies of Gag. In addition, consideration of the relationship between the radial position of different Gag domains and their lateral spacings in particles of different diameters, suggests that the N-terminal MA (matrix) domain does not form a single, regular lattice in immature retrovirus particles.

Avian Sarcoma Viruses↗
PubMed2005

Classification and three-dimensional reconstruction of unevenly distributed or symmetry mismatched features of icosahedral particles.

Methods for the three-dimensional reconstruction of icosahedral particles, such as spherical viruses, from electron micrographs are well established. These methods take advantage of the 60-fold symmetry of the icosahedral group. Several features within these particles, however, may deviate from icosahedral symmetry. Examples include viral genomes, symmetry mismatched vertex proteins, unique DNA packaging vertices, flexible proteins, and proteins that are present at less than 100% occupancy. Such asymmetrically distributed features are smeared in the final density map when icosahedral symmetry is applied. Here, we describe a novel approach to classifying, analysing, and obtaining three-dimensional reconstructions of such features. The approach uses the orientation information derived from the icosahedral orientation search to facilitate multivariate statistical analysis and to limit the orientational degrees of freedom for reconstruction. We demonstrate the application of this approach to images of Kelp fly Virus. In this case, each virion may have two different types of fivefold vertex. We use our approach to produce independent reconstructions of the two types of vertex.

Algorithms↗
PubMed2005

Classification of single-projection reconstructions for cryo-electron microscopy data of icosahedral viruses.

We present a novel strategy for classification of heterogeneous electron microscopy data of icosahedral virus particles. The effectiveness of the procedure, which is based on classification of single-projection reconstructions (SPRs), is first investigated using simulated data. Of several reconstruction approaches examined, best results were obtained with algebraic reconstruction techniques (ART) when providing prior information about the reconstruction in the form of a starting volume. The results presented indicate that SPR-classification is sufficiently sensitive to classify assemblies with differences of only a few percent of the total mass. The usefulness of this procedure is illustrated by application to a heterogeneous cryo-electron microscopy dataset of adenovirus mutant dl313, lacking minor coat protein IX. These data were successfully divided into two distinct classes, in agreement with gel analysis and immuno-electron microscopy results. The classes yielded a wildtype-like reconstruction and a reconstruction representing the polypeptide IX-deficient dl313 virion. As the largest difference between these volumes is found at the location previously assigned to the external portion of minor coat protein polypeptide IIIa, questions arise concerning the current adenovirus model.

Adenoviruses, Human↗
PubMed2004

Insights into assembly from structural analysis of bacteriophage PRD1.

The structure of the membrane-containing bacteriophage PRD1 has been determined by X-ray crystallography at about 4 A resolution. Here we describe the structure and location of proteins P3, P16, P30 and P31. Different structural proteins seem to have specialist roles in controlling virus assembly. The linearly extended P30 appears to nucleate the formation of the icosahedral facets (composed of trimers of the major capsid protein, P3) and acts as a molecular tape-measure, defining the size of the virus and cementing the facets together. Pentamers of P31 form the vertex base, interlocking with subunits of P3 and interacting with the membrane protein P16. The architectural similarities with adenovirus and one of the largest known virus particles PBCV-1 support the notion that the mechanism of assembly of PRD1 is scaleable and applies across the major viral lineage formed by these viruses.

Amino Acid Sequence↗
PubMed2004

The stoichiometry of Gag protein in HIV-1.

The major structural components of HIV-1 are encoded as a single polyprotein, Gag, which is sufficient for virus particle assembly. Initially, Gag forms an approximately spherical shell underlying the membrane of the immature particle. After proteolytic maturation of Gag, the capsid (CA) domain of Gag reforms into a conical shell enclosing the RNA genome. This mature shell contains 1,000-1,500 CA proteins assembled into a hexameric lattice with a spacing of 10 nm. By contrast, little is known about the structure of the immature virus. We used cryo-EM and scanning transmission EM to determine that an average (145 nm diameter) complete immature HIV particle contains approximately 5,000 structural (Gag) proteins, more than twice the number from previous estimates. In the immature virus, Gag forms a hexameric lattice with a spacing of 8.0 nm. Thus, less than half of the CA proteins form the mature core.

Cryoelectron Microscopy↗
PubMed2004

Three-dimensional structures of translating ribosomes by Cryo-EM.

Cryo-electron microscopy and image reconstruction techniques have been used to obtain three-dimensional maps for E. coli ribosomes stalled following translation of three representative proteins. Comparisons of these electron density maps, at resolutions of between 13 and 16 A, with that of a nontranslating ribosome pinpoint specific structural differences in stalled ribosomes and identify additional material, including tRNAs and mRNA. In addition, the tunnel through the large subunit, the anticipated exit route of newly synthesized proteins, is partially occluded in all the stalled ribosome structures. This observation suggests that significant segments of the nascent polypeptide chains examined here could be located within an expanded tunnel, perhaps in a rudimentary globular conformation. Such behavior could be an important aspect of the folding of at least some proteins in the cellular environment.

Cryoelectron Microscopy↗
PubMed2004

Cryoelectron microscopy of mouse mammary tumor virus.

Cryoelectron microscopy of Mouse mammary tumor virus, a Betaretrovirus, provided information about glycoprotein structure and core formation. The virions showed the broad range of diameters typical of retroviruses. Betaretroviruses assemble cytoplasmically, so the broad size range cannot reflect the use of the plasma membrane as a platform for assembly.

Cell Line↗
PubMed2003

Structural organization of authentic, mature HIV-1 virions and cores.

Mature, infectious HIV-1 particles contain a characteristic cone-shaped core that encases the viral RNA and replication proteins. The architectures of mature virions and isolated cores were studied using cryo-electron microscopy. The average size ( approximately 145 nm) of the virion was unchanged during maturation. Most virions contained a single core but roughly one-third contained two or more cores. Consideration of the capsid protein concentration during core assembly indicated that core formation in vivo is template-mediated rather than concentration-driven. Although most cores were conical, 7% were tubular. These displayed a stacked-disc arrangement with 7-, 8-, 9- or 10-fold axial symmetry. Layer line filtration of these images showed that the capsid subunit arrangement is consistent with a 9.6 nm hexamer resembling that previously seen in the helical tubes assembled from purified capsid protein. A common reflection (1/3.2 nm) shared between the tubular and conical cores suggested they share a similar organization. The extraordinary flexibility observed in the assembly of the mature core appears to be well suited to accommodating variation and hence there may be no single structure for the infectious virion.

Cell Line↗
PubMed2003

Depositing electron microscopy maps.

A meeting was held at the European Bioinformatics Institute (EBI) in Hinxton, United Kingdom to discuss recent progress in the development of EMD, a database for maps determined by electron microscopy that is now integrated with MSD, the macromolecular structure database at EBI. This meeting of representatives of many of the major image processing groups in electron microscopy also discussed possible software developments that would ease the documentation and deposition of such datasets. The meeting concluded with a strong endorsement of map deposition in electron microscopy and its linkage with the family of archival databases in biomedical research.

Computational Biology↗
PubMed2003

Do lipid rafts mediate virus assembly and pseudotyping?

Co-infection of a host cell by two unrelated enveloped viruses can lead to the production of pseudotypes: virions containing the genome of one virus but the envelope proteins of both viruses. The selection of components during virus assembly must therefore be flexible enough to allow the incorporation of unrelated viral membrane proteins, yet specific enough to exclude the bulk of host proteins. This apparent contradiction has been termed the pseudotypic paradox. There is mounting evidence that lipid rafts play a role in the assembly pathway of non-icosahedral, enveloped viruses. Viral components are concentrated initially in localized regions of the plasma membrane via their interaction with lipid raft domains. Lateral interactions of viral structural proteins amplify the changes in local lipid composition which in turn enhance the concentration of viral proteins in the rafts. An affinity for lipid rafts may be the common feature of enveloped virus proteins that leads to the formation of pseudotypes.

Animals↗
PubMed2003

The tailless icosahedral membrane virus PRD1 localizes the proteins involved in genome packaging and injection at a unique vertex.

The double-stranded DNA (dsDNA) virus PRD1 carries its genome in a membrane surrounded by an icosahedral protein shell. The shell contains 240 copies of the trimeric P3 protein arranged with a pseudo T = 25 triangulation that is reminiscent of the mammalian adenovirus. DNA packaging and infection are believed to occur through the vertices of the particle. We have used immunolabeling to define the distribution of proteins on the virion surface. Antibodies to protein P3 labeled the entire surface of the virus. Most of the 12 vertices labeled with antibodies directed against proteins P5, P2, and P31. These proteins are known to function in virus binding to the cell surface. Proteins P6, P11, and P20 were found on a single vertex per virion. The P6 and P20 proteins are believed to function in DNA packaging. Protein P11 is a pilot protein that is involved in a complex that mediates the early stages of DNA entry to the host cell. Labeling with antibodies to P5 or P2 did not affect the labeling of P6, the unique vertex protein. Labeling with antibodies to the unique vertex protein P6 interfered with the labeling by antibodies to the unique vertex protein P20. We conclude that PRD1 utilizes 11 of its vertices for initial receptor binding. It utilizes a single, unique vertex for both DNA packing during assembly and DNA delivery during infection.

Bacteriophage PRD1↗
PubMed2002

Minor proteins, mobile arms and membrane-capsid interactions in the bacteriophage PRD1 capsid.

Bacteriophage PRD1 shares many structural and functional similarities with adenovirus. A major difference is the PRD1 internal membrane, which acts in concert with vertex proteins to translocate the phage genome into the host. Multiresolution models of the PRD1 capsid, together with genetic analyses, provide fine details of the molecular interactions associated with particle stability and membrane dynamics. The N- and C-termini of the major coat protein (P3), which are required for capsid assembly, act as conformational switches bridging capsid to membrane and linking P3 trimers. Electrostatic P3-membrane interactions increase virion stability upon DNA packaging. Newly revealed proteins suggest how the metastable vertex works and how the capsid edges are stabilized.

Bacteriophage PRD1↗