Structure of the capsid of pf3 filamentous phage determined from X-ray fibre diffraction data at 3.1 Å resolution

Welsh, Liam; Symmons, Martyn F.; Sturtevant, Julian M.; Marvin, D.A.; Perham, Richard N.

doi:10.1006/jmbi.1998.2081

Cited by 53 publications

(74 citation statements)

References 55 publications

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“…Sample Preparation-Pf1 phage was isolated and purified from infected cultures of P. aeruginosa, strain K, grown in minimal M9 media with 15 NH 4 Cl and [U- 13 C]glucose as the sole nitrogen and carbon sources as described earlier (15). Following density gradient ultracentrifugation, the sample in high CsCl was precipitated with 4% w/v polyethylene glycol (PEG) 8000 (Fluka Biochemicals), pelleted, and redissolved to a concentration of ϳ1 mg/ml in 10 mM Tris, pH 8.4.…”

Section: Methodsmentioning

confidence: 99%

“…To account for the unit stoichiometry, one can consider repeats of 71 dimers in 26 turns, where subunits in the dimer differ in structure slightly because of up-strand versus down-strand interactions (12). For Pf1 H , the apparent repeat is about 7.8 nm, and the symmetry assigned is nominally 27 subunits per 5 turns (or 5.40 subunits/turn), but this symmetry does not account fully for features of some diffraction patterns and NMR data (13)(14)(15). The transition between the two symmetries is estimated to occur between 280 and 286 K depending on pH and salt concentration.…”

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confidence: 99%

“…Based on our prior assignments of the coat protein subunit (15), we have used 13 C homonuclear correlation spectroscopy to map transition-induced perturbations in the assigned NMR chemical shifts. We report on a significant number of such perturbations.…”

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confidence: 99%

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Intersubunit Hydrophobic Interactions in Pf1 Filamentous Phage

Goldbourt

Day²,

McDermott

2010

Journal of Biological Chemistry

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Magic angle spinning solid-state NMR has been used to study the structural changes in the Pf1 filamentous bacteriophage, which occur near 10°C. Comparisons of NMR spectra recorded above and below 10°C reveal reversible perturbations in many NMR chemical shifts, most of which are assigned to atoms of hydrophobic side chains of the 46-residue subunit. The changes mainly involve groups located in patches on the interfaces between neighboring capsid subunits. The observations show that the transition adjusts the hydrophobic interfaces between fairly rigid subunits. The low temperature form has been generally more amenable to structure determination; spin diffusion experiments on this form revealed unambiguous contacts between side chains of neighboring subunits. These contacts are important constraints for structure modeling.

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Intersubunit Hydrophobic Interactions in Pf1 Filamentous Phage

Goldbourt

Day²,

McDermott

2010

Journal of Biological Chemistry

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“…It was proposed that the subunit consists of an N terminus that forms a double hook, a C terminus with an unraveled ␣-helix, and a central portion of three ␣-helices with two bends near the center. An assumed capsid helical symmetry for the high-temperature form of the virion (6,20) was used to generate the intersubunit contacts for a model of the capsid (16,17). In a more recent solid-state NMR study from our group (18,19), magic angle spinning solid-state NMR (MAS SSNMR) was applied to nonaligned hydrated virions in polyethylene glycol precipitates.…”

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confidence: 99%

Conformational dynamics of an intact virus: Order parameters for the coat protein of Pf1 bacteriophage

Lorieau

Day²,

McDermott

2008

Proc. Natl. Acad. Sci. U.S.A.

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This study has examined the atomic-level dynamics of the protein in the capsid of filamentous phage Pf1. This capsid consists of Ϸ7,300 small subunits of only 46 aa in a helical array around a highly extended, circular single-stranded DNA molecule of 7,349 nt. Measurements were made of site-specific, solid-state NMR order parameters, ͗S͘, the values which are dimensionless quantities between 0 (mobile) and 1 (static) that characterize the amplitudes of molecular bond angular motions that are faster than microseconds. It was found that the protein subunit backbone is very static, and of particular interest, it appears to be static at residues glycine 15 and glutamine 16 where it had been previously thought to be mobile. In contrast to the backbone, several side chains display large-amplitude angular motions. Side chains on the virion exterior that interact with solvent are highly mobile, but surprisingly, the side chains of residues arginine 44 and lysine 45 near the DNA deep in the interior of the virion are also highly mobile. The largeamplitude dynamic motion of these positively charged side chains in their interactions with the DNA were not previously expected. The results reveal a highly dynamic aspect of a DNA-protein interface within a virus.solid-state NMR ͉ motion ͉ side chain T he Pf1 bacteriophage is a 2-m-long filamentous virus that infects Pseudomonas aeruginosa strain K (1, 2). The virion consists of a circular single-stranded DNA genome of 7,349 nt (3) within a cylindrical capsid of Ϸ7,300 major coat protein subunits that have 1:1 stoichiometric interactions (4, 5) with nucleotides along the filament, as well as a small number of other proteins interacting with reverse turns of DNA at the ends. A variety of spectroscopic and diffraction studies of Pf1 have been carried out (5-19), including studies by solid-state NMR of the protein in the intact virion (16)(17)(18)(19), as in the present study, and by solution NMR of the isolated protein in detergent micelles (8,14) as a model of its structure in the membrane before virion assembly. Virion assembly occurs at the membrane in such a way that major coat protein subunits are packed in the filament as ␣-helices with their Nterminal regions on the outside and their C-terminal regions buried in the interior to interact with the DNA.One of the studies of the Pf1 virion by solid-state NMR was on magnetically aligned hydrated virions, and it characterized the path and orientation of the backbone of the ␣-helical major capsid protein subunit by means of 1 H-15 N polarization inversion spin exchange at the magic angle (PISEMA) (16,17). It was proposed that the subunit consists of an N terminus that forms a double hook, a C terminus with an unraveled ␣-helix, and a central portion of three ␣-helices with two bends near the center. An assumed capsid helical symmetry for the high-temperature form of the virion (6, 20) was used to generate the intersubunit contacts for a model of the capsid (16,17). In a more recent solid-state NMR study from our group (18, 19), m...

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“…For example, a difference in the packing of the capsid proteins from Pf1 (19) versus Pf3 (27) In our model, the extra 20% mass of HifA is seen as the surface-exposed region that extends furthest from the helical axis (arrow in C), corresponding to regions of highly variable amino acid sequence of the HifA subunit. of 2.4 in their DNA-to-protein ratios.…”

Section: Discussionmentioning

confidence: 92%

Structure and Function of Hib Pili fromHaemophilus influenzaeType b

Bullitt

2002

J Bacteriol

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Pathogenic bacteria are specifically adapted to bind to their customary host. Disease is then caused by subsequent colonization and/or invasion of the local environmental niche. Initial binding of Haemophilus influenzae type b to the human nasopharynx is facilitated by Hib pili, filaments expressed on the bacterial surface. With three-dimensional reconstruction of electron micrograph images, we show that Hib pili comprise a helix 70 Å in diameter with threefold symmetry. The Hib pilus filament has 3.0 subunits per turn, with each set of three subunits translated 26.9 Å along and rotated 53 degrees about the helical axis. Amino acid sequence analysis of pilins from Hib pili and from P-pili expressed on uropathogenic Escherichia coli were used to predict the physical location of the highly variable and immunogenic region of the HifA pilin in the Hib pilus structure. Structural differences between Hib pili and P-pili suggest a difference in the strategies by which bacteria remain bound to their host cells: P-pili were shown to be capable of unwinding to five times their original length (E. Bullitt and L. Makowski, Nature 373:164-167, 1995), while damage to Hib pili occurs by slight shearing of subunits with respect to those further along the helical axis. This capacity to resist unwinding may be important for continued adherence of H. influenzae type b to the nasopharynx, where the three-stranded Hib pilus filaments provide a robust tether to withstand coughs and sneezes.

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Structure of the capsid of pf3 filamentous phage determined from X-ray fibre diffraction data at 3.1 Å resolution

Cited by 53 publications

References 55 publications

Intersubunit Hydrophobic Interactions in Pf1 Filamentous Phage

Intersubunit Hydrophobic Interactions in Pf1 Filamentous Phage

Conformational dynamics of an intact virus: Order parameters for the coat protein of Pf1 bacteriophage

Structure and Function of Hib Pili fromHaemophilus influenzaeType b

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