Donald A. Marvin

Molecular models and structural comparisons of native and mutant class I filamentous bacteriophages : Ff (fd, f1, M13), If1 and IKe

The filamentous bacteriophages are flexible rods about 1 to 2 microns long and 6 nm in diameter, with a helical shell of protein subunits surrounding a DNA core. The approximately 50-residue coat protein subunit is largely alpha-helix and the axis of the alpha-helix makes a small angle with the axis of the virion. The protein shell can be considered in three sections: the outer surface, occupied by the N-terminal region of the subunit, rich in acidic residues that interact with the surrounding solvent and give the virion a low isoelectric point; the interior of the shell, including a 19-residue stretch of apolar side-chains, where protein subunits interact mainly with each other; and the inner surface, occupied by the C-terminal region of the subunit, rich in basic residues that interact with the DNA core. The fact that virtually all protein side-chain interactions are between different subunits in the coat protein array, rather than within subunits, makes this a useful model system for studies of interactions between alpha-helix subunits in a macromolecular assembly. We describe molecular models of the class I filamentous bacteriophages. This class includes strains fd, f1, M13 (these 3 very similar strains are members of the Ff group), If1 and IKe. Our model of fd has been refined to fit quantitative X-ray fibre diffraction data to 30 A resolution in the meridional direction and 7 A resolution in the equatorial direction. A simulated 3.3 A resolution diffraction pattern from this model has the same general distribution of intensity as the experimental diffraction pattern. The observed diffraction data at 7 A resolution are fitted much better by the calculated diffraction pattern of our molecular model than by that of a model in which the alpha-helix subunit is represented by a rod of uniform density. The fact that our fd model explains the fd diffraction data is only part of our structure analysis. The atomic details of the model are supported by non-diffraction data, in part previously published and in part newly reported here. These data include information about permitted or forbidden side-chain replacements, about the effect of chemical modification, and about spectroscopic experiments.(ABSTRACT TRUNCATED AT 400 WORDS)

Adsorption complex of filamentous fd virus

Abstract We have directly visualized what appears to be the adsorption apparatus of a filamentous virus. Electron microscopy reveals at least three knobs, located specifically at one end of the virion, which is more tapered than is the other end. The knobs are attached to the virion tip by connecting stems that are too thin to be seen in most preparations. The protease subtilisin digests away the adsorption protein from the virus, as shown by electrophoretic analysis, and it removes the knobs seen by electron microscopy. Resistance of virion DNAs to endonucleases, even after removal of the terminal knobs with subtilisin, indicates that the thin stems do not consist of DNA. The size of the knobs indicates that each is principally a monomer of the viral adsorption protein; each stem is presumed to be a connecting protein bridge.

A nucleoprotein complex in bacteria infected with Pf1 filamentous virus: Identification and electron microscopic analysis

Abstract We report the discovery, partial purification, and high-resolution electron microscopic characterization of an intracellular complex from Pseudomonas aeruginosa bacteria infected by Pf1 filamentous virus. The Pf1 complex resembles the virion precursor complex of DNA and viral gene 5 protein formed by fd virus of Escherichia coli , but the two complexes differ in structure. Image reconstruction indicates that both complexes are single-start morphological helices; specimen tilting shows the Pf1 helix to be right-handed. Although the Pf1 and fd complexes contain a similar number of nucleotides per axial unit length, the mean distance between helical turns is 61 A for Pf1 but 91 A for fd under the conditions used for our measurements; two turns of the fd nucleoprotein helix contain about as many nucleotides as do three turns of the Pf1 helix. The Pf1 complex is much shorter than are Pf1 virions, in contrast to the similar lengths of the fd virion and complex. The fd complex is extremely flexible, but the Pf1 complex is more highly regular in structure. Most significant, calculations based on our data indicate that the DNA in the Pf1 complex is probably located at a smaller radius than in the bulk of the protein. If the DNA and morphological helices coincide, the DNA in the Pf1 complex must be well inside of (axial to) the outeer protein surfaces of the complex, rather than being wrapped around the protein subunits as proposed by others for fd complex.

Structure of the fd DNA--gene 5 protein complex in solution. A neutron small-angle scattering study.

Neutron small-angle scattering has been used to investigate the fd DNA-gene 5 protein complex in solution. Results are as follows. 1. (1) The mass per unit length is found to be 1380 or 1610 daltons/A, depending upon whether one gene 5 protein molecule is assumed to bind to four or five nucleotides, respectively. These values correspond to axial subunit repeats of 7.9 or 7.0 A and to total contour lengths in solution of 1.27 or 0.90 μm. For a helix of pitch 90 A there are between about 11 and 13 proteins per turn. 2. (2) The cross-sectional radius of gyration at infinite contrast of the complex is 34.5 ± 1 A. 3. (3) The structure must be quite open and solvated as indicated by the dry volume per subunit, the mass per unit length, and the radius of gyration. The volume occupied per subunit in a uniform cylinder having the measured radius of gyration and mass per unit length is about four times greater than the measured subunit dry volume in the complex. 4. (4) From the change with contrast of both the measured radius of gyration and the position of a subsidiary maximum we conclude that the DNA could not be on the outer periphery of the helical structure. This is supported by a calculation of the maximum radius of the DNA backbone. With the possible exception of the positioning of the DNA, our results for the complex in solution are in good agreement with a model proposed by McPherson et al. (1979b) for the complex structure. 5. (5) The complex formed by reconstitution in vitro is not substantially different in its solution structure from the in vivo complex isolated from infected cells.

Pf1 Inovirus. Electron density distribution calculated by a maximum entropy algorithm from native fibre diffraction data to 3 A resolution and single isomorphous replacement data to 5 A resolution.

We have calculated the electron density distribution of the Pf1 strain of filamentous bacteriophage by a maximum entropy method. In the calculation we included native X-ray fibre diffraction data extending to 3 A resolution in the meridional direction on 60 layerlines that are resolved to 4 A in the equatorial direction, and lower resolution data from a single isomorphous derivative iodinated on the Tyr25 residue. The electron density map indicates that the 46-residue protein subunit is a single, gently curved stretch of alpha-helix with its axis at an angle of about 20 degrees to the axis of the virion. The alpha-helix subunit curves around the virion axis by about 1/6 turn, and decreases from about 27 A radius to about 13 A radius in the virion as the amino acid sequence of the subunit runs from the N terminus to the C terminus. Nearest-neighbour alpha-helical subunits are about 10 A apart along their length, and the axis of each subunit makes an unexpected negative angle with its nearest neighbours in the virion. To confirm the validity of the maximum entropy calculation, we have varied the constraints on the calculation. All variations result in either a map that is close to the original map or a map that cannot be interpreted in terms of secondary structure: we find only one map that makes structural sense.

Structure of F-pili: reassessment of the symmetry.

Reassessment of the X-ray fibre diffraction patterns of F-pili using a more accurate subunit molecular weight suggests that subunits in F-pili are related by a fivefold rotation axis around the pilus axis. The identity of this fivefold symmetry with the fivefold rotation axis that relates the subunits in fd bacteriophage supports a simple model for tip-to-tip adsorption of bacteriophage to pili.

The molecular structure and structural transition of the α-helical capsid in filamentous bacteriophage Pf1

The major coat protein in the capsid of Pf1 filamentous bacteriophage (Inovirus) forms a helical assembly of about 7000 identical protein subunits, each of which contains 46 amino-acid residues and can be closely approximated by a single gently curved alpha-helix. Since the viral DNA occupies the core of the tubular capsid and appears to make no significant specific interactions with the capsid proteins, the capsid is a simple model system for the study of the static and dynamic properties of alpha-helix assembly. The capsid undergoes a reversible temperature-induced structural transition at about 283 K between two slightly different helix forms. The two forms can coexist without an intermediate state, consistent with a first-order structural phase transition. The molecular model of the higher temperature form was refined using improved X-ray fibre diffraction data and new refinement and validation methods. The refinement indicates that the two forms are related by a change in the orientation of the capsid subunits within the virion, without a significant change in local conformation of the subunits. On the higher temperature diffraction pattern there is a region of observed intensity that is not consistent with a simple helix of identical subunits; it is proposed that the structure involves groups of three subunits which each have a slightly different orientation within the group. The grouping of subunits suggests that a change in subunit libration frequency could be the basis of the Pf1 structural transition; calculations from the model are used to explore this idea.

On the structures of filamentous bacteriophage Ff (fd, f1, M13)

The filamentous bacteriophage (Inovirus) strain Ff (fd, f1, M13) is widely used in molecular biophysics as a simple model system. A low resolution molecular model of the fd protein coat has been reported, derived from iterative helical real space reconstruction of cryo-electron micrographs (cryoEM). This model is significantly different from the model previously derived from X-ray fibre diffraction and solid-state NMR. We show that the cryoEM model agrees neither with solid-state NMR data nor with X-ray fibre diffraction data of fd, and has some puzzling structural features, for instance nanometre holes through the protein coat. We refine the cryoEM model against the X-ray data, and find that the model after refinement closely approximates the model derived directly from X-ray fibre diffraction and solid-state NMR data. We suggest possible reasons for the differences between the models derived from cryoEM and X-ray diffraction.

Consensus Structure of Pf1 Filamentous Bacteriophage from X-Ray Fibre Diffraction and Solid-State NMR.

Filamentous bacteriophages (filamentous bacterial viruses or Inovirus) are simple and well-characterised macromolecular assemblies that are widely used in molecular biology and biophysics, both as paradigms for studying basic biological questions and as practical tools in areas as diverse as immunology and solid-state physics. The strains fd, M13 and f1 are virtually identical filamentous phages that infect bacteria expressing F-pili, and are sometimes grouped as the Ff phages. For historical reasons fd has often been used for structural studies, but M13 and f1 are more often used for biological experiments. Many other strains have been identified that are genetically quite distinct from Ff and yet have a similar molecular structure and life cycle. One of these, Pf1, gives the highest resolution X-ray fibre diffraction patterns known for filamentous bacteriophage. These diffraction patterns have been used in the past to derive a molecular model for the structure of the phage. Solid-state NMR experiments have been used in separate studies to derive a significantly different model of Pf1. Here we combine previously published X-ray fibre diffraction data and solid-state NMR data to give a consensus structure model for Pf1 filamentous bacteriophage, and we discuss the implications of this model for assembly of the phage at the bacterial membrane.

Two Forms of Pf1 Inovirus: X-Ray Diffraction Studies on a Structural Phase Transition and a Calculated Libration Normal Mode of the Asymmetric Unit

Inovirus is a helical array of α-helical protein asymmetric units surrounding a DNA core. X-ray fibre diffraction studies show that the Pf1 species of Inovirus can undergo a reversible temperature-induced transition between two similar structural forms having slightly different virion helix parameters. Molecular models of the two forms show no evidence for altered interactions between the protein and either the solvent or the viral DNA; but there are significant differences in the shape and orientation of the protein asymmetric unit, related to the changes in the virion parameters. Normal modes involving libration of whole asymmetric units are in a frequency range with appreciable entropy of libration, and the structural transition may be related to changes in libration.