Yizhi Tao

Assembly of a Tailed Bacterial Virus and Its Genome Release Studied in Three Dimensions

We present the first three-dimensional reconstruction of a prolate, tailed phage, and its empty prohead precursor by cryo-electron microscopy. The head-tail connector, the central component of the DNA packaging machine, is visualized for the first time in situ within the Bacillus subtilis dsDNA phage phi29. The connector, with 12- or 13-fold symmetry, appears to fit loosely into a pentameric vertex of the head, a symmetry mismatch that may be required to rotate the connector to package DNA. The prolate head of phi29 has 10 hexameric units in its cylindrical equatorial region, and 11 pentameric and 20 hexameric units comprise icosahedral end-caps with T=3 quasi-symmetry. Reconstruction of an emptied phage particle shows that the connector and neck/tail assembly undergo significant conformational changes upon ejection of DNA.

Structure of bacteriophage T4 fibritin: a segmented coiled coil and the role of the C-terminal domain.

BACKGROUND Oligomeric coiled-coil motifs are found in numerous protein structures; among them is fibritin, a structural protein of bacteriophage T4, which belongs to a class of chaperones that catalyze a specific phage-assembly process. Fibritin promotes the assembly of the long tail fibers and their subsequent attachment to the tail baseplate; it is also a sensing device that controls the retraction of the long tail fibers in adverse environments and, thus, prevents infection. The structure of fibritin had been predicted from sequence and biochemical analyses to be mainly a triple-helical coiled coil. The determination of its structure at atomic resolution was expected to give insights into the assembly process and biological function of fibritin, and the properties of modified coiled-coil structures in general. RESULTS The three-dimensional structure of fibritin E, a deletion mutant of wild-type fibritin, was determined to 2.2 A resolution by X-ray crystallography. Three identical subunits of 119 amino acid residues form a trimeric parallel coiled-coil domain and a small globular C-terminal domain about a crystallographic threefold axis. The coiled-coil domain is divided into three segments that are separated by insertion loops. The C-terminal domain, which consists of 30 residues from each subunit, contains a beta-propeller-like structure with a hydrophobic interior. CONCLUSIONS The residues within the C-terminal domain make extensive hydrophobic and some polar intersubunit interactions. This is consistent with the C-terminal domain being important for the correct assembly of fibritin, as shown earlier by mutational studies. Tight interactions between the C-terminal residues of adjacent subunits counteract the latent instability that is suggested by the structural properties of the coiled-coil segments. Trimerization is likely to begin with the formation of the C-terminal domain which subsequently initiates the assembly of the coiled coil. The interplay between the stabilizing effect of the C-terminal domain and the labile coiled-coil domain may be essential for the fibritin function and for the correct functioning of many other alpha-fibrous proteins.

Structure determination of the head–tail connector of bacteriophage φ29

The head-tail connector of bacteriophage phi29 is composed of 12 36 kDa subunits with 12-fold symmetry. It is the central component of a rotary motor that packages the genomic dsDNA into preformed proheads. This motor consists of the head-tail connector, surrounded by a phi29-encoded, 174-base, RNA and a viral ATPase protein, both of which have fivefold symmetry in three-dimensional cryo-electron microscopy reconstructions. DNA is translocated into the prohead through a 36 A diameter pore in the center of the connector, where the DNA takes the role of a motor spindle. The helical nature of the DNA allows the rotational action of the connector to be transformed into a linear translation of the DNA. The crystal structure determination of connector crystals in space group C2 was initiated by molecular replacement, using an approximately 20 A resolution model derived from cryo-electron microscopy. The model phases were extended to 3.5 A resolution using 12-fold non-crystallographic symmetry averaging and solvent flattening. Although this electron density was not interpretable, the phases were adequate to locate the position of 24 mercury sites of a thimerosal heavy-atom derivative. The resultant 3.2 A single isomorphous replacement phases were improved using density modification, producing an interpretable electron-density map. The crystallographically refined structure was used as a molecular-replacement model to solve the structures of two other crystal forms of the connector molecule. One of these was in the same space group and almost isomorphous, whereas the other was in space group P2(1)2(1)2. The structural differences between the oligomeric connector molecules in the three crystal forms and between different monomers within each crystal show that the structure is relatively flexible, particularly in the protruding domain at the wide end of the connector. This domain probably acts as a bearing, allowing the connector to rotate within the pentagonal portal of the prohead during DNA packaging.

Crystal structure of the human astrovirus capsid spike

Astroviruses are single-stranded, plus-sense RNA viruses that infect both mammals and birds, causing gastroenteritis and other extraintestinal diseases. Clinical studies have established astroviruses as the second leading cause of viral diarrhea in young children. Here we report the crystal structure of the human astrovirus dimeric surface spike determined to 1.8-Å resolution. The overall structure of each spike/projection domain has a unique three-layered β-sandwiches fold, with a core, six-stranded β-barrel structure that is also found in the hepatitis E virus capsid protrusions, suggesting a closer phylogenetic relationship between these two viruses than previously acknowledged. Based on a hepatitis E virus capsid model, we performed homology modeling and produced a complete, T = 3 astrovirus capsid model with features remarkably similar to those observed in a cryoelectron microscopy reconstruction image of a human astrovirus. Mapping conserved residues onto the astrovirus projection domain revealed a putative receptor binding site with amino acid compositions characteristic for polysaccharide recognition. Our results will have an important impact on future characterization of astrovirus structure and function, and will likely have practical applications in the development of vaccines and antivirals.

The ins and outs of four-tunneled Reoviridae RNA-dependent RNA polymerases.

RNA-dependent RNA polymerases (RdRps) of the segmented double-stranded (ds) RNA viruses of the Reoviridae family exhibit distinguishing structural elements, enabling the enzymes to function within the confines of a proteinaceous core particle. These globular, cage-like polymerases are traversed by four well-defined tunnels, which not only allow template RNAs, nucleotides, and divalent cations to access the interior catalytic site, but also provide two distinct exit conduits for RNA templates and products--one leading out of the core and the other back inside the core. Although Reoviridae RdRps are intrinsically capable of binding template, their catalytic activities are tightly regulated by interactions with core shell proteins. This intra-particle mechanism of RNA synthesis coordinates genome packaging with replication during the infectious cycle.

Structure of Bacteriophage T4 Fibritin M: a Troublesome Packing Arrangement

Fibritin, a 52 kDa product of bacteriophage T4 gene wac, forms 530 A long fibers, named whiskers, that attach to the phage neck and perform a helper function during phage assembly. Fibritin is a homotrimer, with its predominant central domain consisting of 12 consecutive alpha-helical coiled-coil segments linked together by loops. The central domain is flanked by small globular domains at both ends. Fibritin M is a genetically engineered fragment of the wild type and contains 74 amino-acid residues corresponding to the last coiled-coil segment and the complete carboxy-terminal domain. The crystals of fibritin M belong to the rare space group P3 with three crystallographically independent trimers in the unit cell. The structure has been established at 1.85 A resolution by combining molecular and isomorphous replacement techniques. One of the two heavy-atom derivatives used was gaseous xenon. A substantial fraction of residues in each independent trimer is disordered to various extents in proportion to the lack of restraints on the molecules provided by the lattice contacts. Accurate modeling of the solvent present in the crystals was crucial for achieving good agreement with experimental data.

Recent developments in cryo-electron microscopy reconstruction of single particles

Cryo-electron microscopy and single-particle 3D image reconstruction techniques have been used to examine a broad spectrum of samples ranging from 500 kDa protein complexes to large subcellular organelles. The attainable resolution has improved rapidly over the past few years. Structures of both symmetric and asymmetric assemblies at approximately 7.5 A have been reported. Together with X-ray crystallography, three-dimensional cryo-electron microscopy reconstruction has provided important insights into the function of many biological systems in their native biochemical contexts.

Structural insight into insect viruses

The first structure of an insect picorna-(small RNA-containing) virus is now available. Although there is considerable similarity in the structures of mammalian and insect picornaviruses, there are also remarkable differences, the most noteworthy being associated with the small, internal, functionally essential, VP4 protein.

Courageous science: structural studies of bluetongue virus core

The structure of the bluetongue virus core was recently reported and represents the largest structure determined to atomic resolution. As a biological machine capable of RNA transcription, the structure has immense biological significance.

Erratum: Detailed architecture of a DNA translocating machine: The high-resolution structure of the bacteriophage φ29 connector particle (Journal of Molecular Biology (2002))

The recent paper by Guasch et al.1 describing the structure of the ϕ29 bacteriophage head–tail connector at 2.1 A resolution (PDB code 1h5w), makes reference to the original 3.2 A resolution structure (PDB code 1fou) published by Simpson et al.2 and used by Guasch et al. to solve the 1h5w structure by molecular replacement. The Guasch paper states that there are marked differences between the 1h5w and 1fou structures in the chain tracing and the side-chain positioning in the wide domain. As these and a number of other statements in the Guasch paper may have led to the impression that these differences are larger than they really are, we give here Tables summarizing the similarities and differences (Tables 1 and ​and22). Table 1 Statistical comparison of the ϕ29 connector wide domains in different structures Table 2 Superposition of the entire monomers in the four reported structures Another paper by Simpson et al.,3 which was accidentally omitted from the text of the Guasch et al. paper, reports two additional structures of the ϕ29 connector: at 3.2 A resolution and at 2.9 A resolution (PDB codes 1jnb and 1ijg, respectively). The 2.9 A resolution structure shows no significant differences from the structure subsequently reported by Guasch et al. (Figure 1; Tables 1 and ​and22). Figure 1 Stereo diagram showing the superposition of the Cα back-bone of monomer A in the 2.1 A resolution structure 1h5w (green) and the 2.9 A resolution 1ijg structure (red).