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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.

Filamentous bacteriophage (Inovirus) is a widely studied model system in molecular biophysics. The structure of the virion has been analysed by various methods, but the methods have seldom questioned the hand of the virion helix. The hand of the helix relating the protein subunits in the class II virus strain Pf1 was chosen by calculating an electron-density distribution from X-ray fibre diffraction data, using a maximum-entropy method, but to our knowledge this method has not been used for a similar purpose in any other system. Moreover, this same hand was extended only by analogy, with no direct analysis of the corresponding data, to the class I virus strain Ff (fd, f1, M13), which has a different helix symmetry. Here we use published solid-state NMR data to confirm the validity of the hand of Pf1 chosen by the maximum-entropy method, and to confirm the extension to Ff.

Daptomycin, a cyclic anionic lipopeptide antibiotic, whose three-dimensional structure was recently solved using solution state NMR (Ball et al. 2004; Jung et al. 2004; Rotondi and Gierasch 2005), requires calcium for function. To date, the exact nature of the interaction between divalent cations, such as Ca(2+) or Mg(2+), has not been fully characterized. It has, however, been suggested that addition of Ca(2+) to daptomycin in a 1:1 molar ratio induces aggregation. Moreover, it has been suggested that certain residues, e.g. Asp3 and Asp7, which are essential for activity (Grunewald et al. 2004; Kopp et al. 2006), may also be important for Ca(2+) binding (Jung et al. 2004). In this work, we have tried: (1) to further pinpoint how Ca(2+) affects daptomycin structure/oligomerization using analytical ultracentrifugation; and (2) to determine whether a specific calcium binding site exists, based on one-dimensional (13)C NMR spectra and molecular dynamics (MD) simulations. The centrifugation results indicated that daptomycin formed micelles of between 14 and 16 monomers in the presence of a 1:1 molar ratio of Ca(2+) and daptomycin. The (13)C NMR data indicated that addition of calcium had a significant effect on the Trp1 and Kyn13 residues, indicating that either calcium binds in this region or that these residues may be important for oligomerization. Finally, the molecular dynamics simulation results indicated that the conformational change of daptomycin upon calcium binding might not be as significant as originally proposed. Similar studies on the divalent cation Mg(2+) are also presented. The implication of these results for the biological function of daptomycin is discussed.

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.