Ions trapped at the interior of gp26 trimerization interface. (a) Worm representation of tail needle gp26, with the calcium and chlorine ions displayed as a gold sphere and orange sphere, respectively. (b) Sequence alignment of the putative tail needle sequence from 12 P22-like phages. For simplicity, only the amino sequence spanning residues 60–103 is displayed. Invariant residues interacting with the two ions are highlighted in dark pink, and partially conserved interacting residues in light pink. Other conserved residues are indicated below the alignment, where an asterisk (*) indicates an invariant residue, and a colon (:) a highly conserved residue. (c, d) Close-up view of the calcium and chloride ions observed at the gp26 internal trimerization interface. In both panels, the green density around the ions represents a F_Obs − F_Cal difference map computed using structure factor amplitudes from the final refined model missing the Ca1 and Cl1 ions. The density was computed to 2.5 Å and contoured at 5σ. The calcium site has a refined B-factor of ∼21 Ų and the chlorine site ∼28 Ų, respectively. The blue density around the ions represents an anomalous map computed using anomalous differences measured at low energy (λ = 1.75 Å, dataset Xe-Low in Table I) and contoured at 2σ.

Xenon gas probes the flexibility of tail needle gp26. (a, b) The structure of gp26 bound to xenon gas was fit into the cryo-EM reconstructions of P22 mature viron determined by Lander et al.9 (a) and Chang et al.8 (b). In both panels (a, b) the seven xenon atoms, the calcium ion, and the chloride ion bound to gp26 are shown as green, gold, and orange spheres, respectively. The C-terminal tip of gp26 has poor density in the cryo-EM reconstructions, likely indicating high flexibility or multiple conformations. In contrast, the two ion traps (gold/orange spheres) are found in the more rigid areas of the protein, characterized by strong density in both cryo-EM reconstructions. (c) Space filling model of gp26, indicating the flexibility observed in the crystal structure and supported by the EM reconstructions. Domains III and IV of the tail needle are flexibly connected the helical core Domain II, which can bend ∼18° relative to the rigid helical core, thus generating a putative scanning device that interacts with the host cell envelope.

Three-dimensional structure of phage P22 tail needle bound to xenon gas. (a) Worm representation of tail needle gp26 bound to seven xenon atoms, one calcium, and one chloride atom (represented as green, gold, orange spheres, respectively). The structure was determined to 1.98 Å resolution and refined to an R_free of 26.8% (Table I). The tail needle is ∼240 Å homotrimer, consisting of four structural domains (indicated by horizontal arrows). All xenon and chloride atoms were found in cavities at the gp26 internal trimerization interface. (b–d) Close-up view of the three classes of xenon sites observed inside gp26 core: the orientation of gp26 shown is perpendicular to that in (a). Gp26 residues lining the binding site are depicted as ball-and-sticks with nitrogen and oxygen atoms colored in blue and red, respectively. Only residues of protomer A of gp26 are labeled. The blue and red densities around xenon atoms represent anomalous difference maps computed using X-ray data measured at low energy (λ = 1.75 Å) and high energy (λ = 0.95 Å), respectively (datasets Xe-Low and Xe-High in Table I). All anomalous maps were computed to 2.5 Å resolution and contoured at 15σ above background. The refined B-factor to 1.98 Å resolution for xenon atoms Xe2, Xe3, Xe5, and Xe6 ranges between 32 and 39 Ų, whereas Xe1, Xe4, and Xe7 have significantly higher B-factor, between 60 and 96 Ų (Table I).

Organization of internal cavities in tail needle gp26. (a) Slice-view of the gp26 space-filling model. Xenon atoms are represented by green spheres, calcium by a gold sphere, chlorine by an orange sphere, and waters as blue spheres. Seven major cavities are detected at the trimeric binding interface using the program, VOIDOO25 all filled by xenon atoms in our crystal structure. The two terminal cavities filled by Xe1 and Xe7 and shaded in pink are “invaginations,” which are defined as cavities in partial exchange with the exterior of protein. Cavities occupied by Xe2, Xe3, Xe4, Xe5, and Xe6 and shaded in white are “voids” completely closed off from the exterior of the protein. (b) Representation of the four cavities clustered at the boundary between gp26 helical Domain II and the triple β-helix, Domain III. The gp26 backbone is represented as a grey tube, with the flexible β-helix highlighted in yellow, and the volume of cavities computed by VOIDOO25 shown as a blue mesh.