The In Situ Structure of T-Series T1 Reveals a Conserved Lambda-Like Tail Tip

Abstract

It is estimated that over 60% of known tailed phages are siphophages, which are characterized by a long, flexible, and non-contractile tail. Nevertheless, entire high-resolution structures of siphophages remain scarce. Using cryo-EM, we resolved the structures of T-series siphophage T1, encompassing its head, connector complex, tail tube, and tail tip, at near-atomic resolution. The density maps enabled us to build the atomic models for the majority of T1 proteins. The T1 head comprises 415 copies of the major capsid protein gp47, arranged into an icosahedron with a triangulation number of seven, decorated with 80 homologous trimers and 60 heterotrimers along the threefold and quasi-threefold axes of the icosahedron. The T1 connector complex is composed of two dodecamers (a portal and an adaptor) and two hexamers (a stopper and a tail terminator). The flexible tail tube comprises approximately 34 hexameric rings of tail tube. The extensive disulfide bond network along the successive tail rings may mediate the flexible bending. The distal tip of T1, which is cone-shaped and assembled by proteins gp33, gp34, gp36, gp37, and gp38, displays structural similarity to that of phage lambda. In conjunction with previous studies of lambda-like siphophages, our structure will facilitate further exploration of the structural and mechanistic aspects of lambda-like siphophages.

Keywords: T-series phages; cone-shaped tail tip; cryo-EM; disulfide bond; lambda-like siphophages.

Figure 1

Overall structure of siphophage T1. (A,B) Side (A) and cut-open (B) views of the asymmetric structure of T1. L₁ and L₃₄ denote the initial (first) and final (thirty-fourth) layers of the tail tube, respectively. The color code is applied to panels (A–C,E). (C) Side view of the ribbon models of connector complex. (D) Zoomed-in view of a particle image of T1. The possible TTP rings are labeled by blue lines. (E) Cut-open view of the ribbon models of the cone-shaped tail tip and a tail tube ring.

Figure 2

Structures of MCPs and CPs of T1. (A) Density maps of a hexon (light steel blue), a penton (olive), and six trimeric CPs comprising four homotrimers and two heterotrimers. The CP gp48 and gp49 are colored in hot pink and slate blue, respectively. (B) Ribbon model of the MCP gp47 shown in four domains. (C,D) Top view of the ribbon models of the heterotrimeric CP (C) and homotrimeric CP (D). (E) Density map (transparency) of CP gp48 superimposed on the ribbon model and C-terminus of gp48 modeled using AlphaFold3. (F) Structural comparison between gp49 (slate blue) and the N-terminus of gp48 (pink) revealing the obvious topological similarity. (G) Interactions at the capsomere–CP interface around the threefold axis (left) and quasi-threefold axis (right) of the icosahedral head. The insets show the zoomed-in views of the structural difference of the N-terminus between gp48 and gp49 interacting with the capsomeres.

Figure 3

Structures of the connector complex. (A) Side view (left) of the density maps and slab view (right) of the ribbon models of the connector complex. The insets show the zoomed-in views of the interactions among the portal–adaptor and the adaptor–stopper. The color coding is identical to that used in Figure 1A. (B–D) Ribbon models of the portal protein gp52 (B), the adaptor protein gp45 (C), and the stopper protein gp44 (D), colored according to their domains. (E) Ribbon model of the tail terminator protein gp42 shown in rainbow colors.

Figure 4

Structure of the tail tube. (A) Side view of four layers of hexameric tail tube (cornflower blue). Only one monomer of tail tube protein gp41 is colored according to its domains. (B) Ribbon model of TTP gp41, colored according to its domains. (C) Cut-open view of the electrostatic potential of the inner surfaces of tail tube. The electrostatic potential scale is shown in the color bar. (D) Zoomed-in view of the inter-ring interactions by the disulfide bond.

Figure 5

Structure of the tail tip. (A) Cut-open (left) and side (right) views of the density maps of the tail tip without the lateral fibers. The inset shows a zoomed-in view of the interactions among the distal tail protein gp37, center fiber protein gp33, and hub protein 36. The color coding is identical to that used in Figure 1A. (B) Ribbon model of the distal tail protein gp37, colored according to its domains. (C) Top view of the interactions between the threefold central fiber protein gp33 and the threefold hub protein gp36. (D) Ribbon models of the center fiber protein gp33. (E) Ribbon models of hub protein 36 and zoomed-in view of the iron–sulfur cluster (green) superimposed on its density map (transparent). (F) Interactions between the trimeric TMP gp38 (gray) and the threefold insert protein gp34 (blue violet).

Figure 6

Structural comparison of tail tip proteins in different siphophages. (A,B) Cut-open views of ribbon models of the tail tips in T1 (A) and lambda ((B), PDB ID: 8k35 and 8xck). (C) Structural comparisons of ribbon models of all tail tip proteins between T1 and lambda, revealing the obvious topological similarity. The color coding is identical to that used in (A,B). (D) Structural comparisons of ribbon models of the tip (the central fiber protein and/or hub protein) among lambda-like siphophages, including T1, lambda (PDB ID: 8k35 and 8xck), Chi (PDB ID: 8VJH), T5 (PDB ID: 7zhj), and DT57C (PDB ID: 8hqz).