José L. Carrascosa

The bacteriophage φ29 head–tail connector imaged at high resolution with the atomic force microscope in buffer solution

The surfaces of two‐ and three‐dimensional φ29 connector crystals were imaged in buffer solution by atomic force microscopy (AFM). Both topographies show a rectangular unit cell with dimensions of 16.5 nm×16.5 nm. High resolution images of connectors from the two‐dimensional crystal surface show two connectors per unit cell confirming the p4212 symmetry. The height of the connector was estimated to be at least 7.6 nm, a value close to that found in previous studies using different techniques. The 12 subunits of the wide connector domain were clearly resolved and showed a right‐handed vorticity. The channel running along the connector had a diameter of 3.7 nm in the wide domain, while it was 1.7 nm in the narrow domain end, thus suggesting a tronco‐conical channel shape. Moreover, the narrow connector end appears to be rather flexible. When the force applied to the stylus was between 50 and 100 pN, the connector end was fully extended. At forces of ∼150 pN, these ends were pushed towards the crystal surface. The complementation of the AFM data with the three‐dimensional reconstruction obtained from electron microscopy not only confirmed the model proposed, but also offers new insights that may help to explain the role of the connector in DNA packing.

Template location in noisy pictures

Abstract This work describes several methods to perform template location in noisy images (specially in very noisy ones), as well as tests to compare their respective speed and accuracy. Three kinds of images have been considered: pictures with additive noise, pictures with multiplicative noise, and real images from electron microscopy. The performances of the different methods have been evaluated and the results obtained are presented.

Structure of phage Φ29 connector protein assembled in vivo

Abstract The protein p10 that forms the connector of phage φ29, has been produced in Escherichia coli harboring a plasmid that carried the gene coding for this protein. The connector protein is assembled in a 13.4-S oligomer that has an apparent molecular weight of 460,000, suggesting that it is a dodecamer. The purified oligomers have been studied by electron microscopy of the isolated particles as well as by image-processing techniques (Fourier and rotational filtering) of artificially induced two-dimensional aggregates. The results show that the purified p10 is assembled in a circular structure with a hole in its center and 12 morphological units in the periphery. Both the morphology and the dimensions of this p10 oligomer are very similar to those of the upper neck collar extracted from φ29 viral particles. The results strongly suggest the close relationship between the p10 oligomers assembled in E. coli and the ones produced in φ29, infected Bacillis subtilis.

A precursor of the neck appendage of Bacillus subtilis phage ø29

Comision Asesora para la Investigacion Cientifica y Fondo Nacional para la Formacion de Personal Investigador

Structural organization of Bacillus subtilis phage φ29. A model

Phage phi29 is a nonisometric virus producing several types of morphological variants in normal infections. The study of these variants by electron microscopy, and their comparison with those from T-even phages, suggest that the capsid of phage phi29 is a prolate icosahedron. Phage phi29 capsid consists of a major protein, p8, and an additional protein, p8.5, making up the fibers. We have determined the number of subunits of each structural protein per viral particle taking into account the phage molecular weight (between 28 and 29.6 x 10(6)), the molecular weight of each structural protein, and the mass percentage of each protein with respect to the total protein mass of the phage. These values, together with the results obtained from chemical crosslinking of the structural proteins on the phage, suggest that the capsid contains protein p8 dimers clustered in trimers.

Conformational changes in bacteriophage Ø 29 connector prevents DNA-binding activity

In vitro DNA packaging activity in a defined system derived from bacteriophage phi 29 depends upon the chemical integrity of the connector protein p10. Proteolytic cleavage of p10 rendered the proheads inactive for DNA packaging. A similar treatment on isolated connectors abolished the DNA-binding activity of the native p10, but the general shape and size of the connector was not changed as revealed by electron microscopy. Analytical ultracentrifugation showed that the proteolyzed connectors had a smaller sedimentation coefficient, while amino acid analysis after dialysis of the proteolyzed p10 confirmed the loss of 16 and 19 amino acids from the amino and carboxy termini, respectively. Low angle X-ray scattering revealed that proteolysis was followed by a small decrease in the radius of gyration and a reorganization of the distal domain of the cylindrical inner part of the connector. Characterization of the cleavage sites in the primary sequence allowed us to propose the location of the DNA-binding domain in the connector model.

A protein similar to Escherichia coli gro EL is present in Bacillus subtilis

Abstract We have found in Bacillus subtilis an oligomer that resembles closely the gro EL oligomer from Escherichia coli. These structures have similar morphology, dimensions and sedimentation coefficients, and both are based on a 65,000 Mr polypeptide. Furthermore, the oligomer from B. subtilis infected with phage φ29 copurifies with a viral protein (p10) that is involved in the early steps of phage head morphogenesis.

Study of two related configurations of the neck of bacteriophage Ø29

Abstract The 3-D reconstructed images of two related viral structures, obtained by digital image processing from electron micrographs, have been compared to obtain insight into their morphological differences. The viral structures that were studied are the proteins that connect the head to the tail of bacteriophage O29. One of these proteins (p10) is assembled as a dodecamer and, once the DNA has been encapsidated into the viral head, this protein interacts with another viral protein (p11) rendering the viral neck. Biochemical analysis by controlled proteolysis revealed a conformational difference in the structure of p10, depending on whether it is assembled in the connector or in the neck complex. Further structural analysis was carried out by digital image processing and computer graphic techniques. The reconstructed density maps were visualized as solid surface representations to reveal gross morphology, and as translucent models to study the inner details of their structure. The use of different density thresholds also allowed us to distinguish interesting morphological features. A channel present in the connector is closed in the final neck. This closing can be correlated with a rearrangement of the subunits of the connector, probably induced by the interaction of protein p11 with p10.

Structures of the Gβ-CCT and PhLP1–Gβ-CCT complexes reveal a mechanism for G-protein β-subunit folding and Gβγ dimer assembly

Significance G-protein signaling contributes to nearly all aspects of cell physiology. In order for signaling to occur, the G-protein αβγ heterotrimer must be assembled from its individual subunits. The G-protein βγ subcomplex does not assemble spontaneously, but requires the protein-folding chaperone cytosolic chaperonin containing TCP-1 (CCT) and its cochaperone phosducin-like protein 1 (PhLP1). To understand the molecular mechanism underlying this process, we determined the structures of two key intermediates in Gβγ assembly, Gβ-CCT and PhLP1–Gβ-CCT, using cryo-electron microscopy and chemical cross-linking. These structures reveal the molecular basis for chaperone-mediated Gβγ assembly that can be exploited to design novel therapeutics to regulate G-protein signaling. G-protein signaling depends on the ability of the individual subunits of the G-protein heterotrimer to assemble into a functional complex. Formation of the G-protein βγ (Gβγ) dimer is particularly challenging because it is an obligate dimer in which the individual subunits are unstable on their own. Recent studies have revealed an intricate chaperone system that brings Gβ and Gγ together. This system includes cytosolic chaperonin containing TCP-1 (CCT; also called TRiC) and its cochaperone phosducin-like protein 1 (PhLP1). Two key intermediates in the Gβγ assembly process, the Gβ-CCT and the PhLP1–Gβ-CCT complexes, were isolated and analyzed by a hybrid structural approach using cryo-electron microscopy, chemical cross-linking coupled with mass spectrometry, and unnatural amino acid cross-linking. The structures show that Gβ interacts with CCT in a near-native state through interactions of the Gγ-binding region of Gβ with the CCTγ subunit. PhLP1 binding stabilizes the Gβ fold, disrupting interactions with CCT and releasing a PhLP1–Gβ dimer for assembly with Gγ. This view provides unique insight into the interplay between CCT and a cochaperone to orchestrate the folding of a protein substrate.

Structural characterization of T7 tail machinery reveals a conserved tubular structure among other Podoviridae family members and suggests a common mechanism for DNA delivery

Bacteriophage tail complexes play an essential role in host recognition and DNA delivery during virus infection. These molecular machines are composed of a tubular structure surrounded by fibers, with a central channel that acts as a conduit for DNA ejection. The T7 tail complex is formed by four proteins: connector (gp8), gatekeeper (gp11), nozzle (gp12), and fibers (gp17). Previous biochemical and structural studies allowed definition of the stoichiometry and order of assembly of these proteins. Here we compared the tail complex from other Podoviridae phages that infect bacteria with Gram− type envelopes (K1E, P-SSP7, and ε15), and found strong similarities with the T7 nozzle; this was supported by sequence alignment and secondary structure prediction studies. These similarities were not observed in the new reconstruction of protein p9 presented here, which builds the hexameric nozzle of ϕ29, a virus that infects Gram+ bacteria. The results suggest that the Podoviridae nozzle has evolved to adapt to membrane composition of the infected host.