Pf1 filamentous bacteriophage: refinement of a molecular model by simulated annealing using 3.3 Å resolution X-ray fibre diffraction data

Abstract

The filamentous bacteriophage Pf1 is structurally similar to the well known Ff (fd, fl, M13) strains, but it gives much better X-ray diffraction patterns, enabling a more detailed analysis of the molecular structure. The 46-residue protein subunit can be closely approximated by a single gently curved stretch of alpha-helix. The axes of the subunits are at a small angle to the virion axis, and several thousand subunits form an overlapping inter-digitated helical array surrounding a DNA core. We have derived a detailed model of the virion based on X-ray data and stereochemical constraints. We have considered potential sources of error in the diffraction data, and used the improved data to study regions where the protein subunit of Pf1 may deviate from a continuous alpha-helix. We use simulated annealing to escape from local minima, and various kinds of electron-density maps to guide the model building. Refinement of the model shows that the first few residues at the N terminus are non-helical, and there is a slight discontinuity in the alpha-helix near the middle of the sequence. The model is consistent both with general structural principles derived from high-resolution analysis of other proteins, and with specific chemical and spectroscopic data about Pf1. We apply the same refinement techniques to an alternative model with a non-helical surface loop between residues 13 and 19. Comparative analysis of models with and without a loop shows that the loop model is not supported by 3.3 A resolution X-ray diffraction data.

Heterogeneous Seeding of a Prion Structure by a Generic Amyloid Form of the Fungal Prion-forming Domain HET-s(218–289)

“…Quantitative equatorial comparisons were made by calculating correlation coefficients between each pair of plots. Correlation between the pH 2.0 equators (correlation coefficient ϭ 0.91) is high (28), and together with the meridional data, indicates that these specimens share the same overall architecture, although the disorientation and low resolution of the data preclude comparisons of atomic structures. Correlation between pH 7.5 and pH 2.0 citric acid or phosphoric acid equatorial plots is low (0.68 and 0.48, respectively), and taken together with the presence or absence of 9.4 Å meridional reflec- tions, clearly demonstrates that the fibrils formed at different pH values have distinct architectures.…”

“…Patterns were compared by calculating correlation coefficients; these coefficients have been shown to be a reliable measure of structural similarity (28). For solenoidal patterns from different samples, made in the same way but at different times and measured at different synchrotrons, correlation coefficients were between 0.98 and 1.00.…”

Fungal prion HET-s as a model for structural complexity and self-propagation in prions

“…The resolution range used for comparisons to WT was ∼33-34 Å (0.03-0.25 Å −1 ), providing comparisons of architecture up to residue-level resolution, whereas comparisons with the stacked β-sheet model were performed at ∼33-35 Å (0.03-0.20Å −1 ), providing comparisons of the main chain. For the wide resolution range compared, the CC is a superior measure of agreement to the R factor because it is unaffected by intensity scaling difficulties caused by solventcontrast effects (43). Correlation coefficients were calculated from background-subtracted equatorial plots as previously described (28).…”

Structure of the Coat Protein in Pf1 Bacteriophage Determined by Solid-state NMR Spectroscopy

“…Although it was well established that most of the coat protein is helical in the bacteriophage particles, the structure of the N-terminal portion was less certain, having been variously described as either helical (PDB entry 1IFN) 5,8,29 or unstructured (PDB entry 2IFN) 5,30 from poorly defined intensity in fiber diffraction electron density maps. 30 The marked deviation of the structure of the N-terminal region from a continuous helix is illustrated in Figure 1(c).…”