Overall structures of phi92 in its two distinct states

For the collection of cryo-EM data, phage phi92 was purified from the E.coli BL21 strain (S1A Fig) and contracted phi92 was produced by treating extended phi92 particles with 3M urea (S1B Fig). A total of 32,356 extended and 9,796 contracted particles were extracted from the native sample and the urea-treated sample, respectively. Icosahedral reconstruction method [31] was utilized to obtain an icosahedral head structure of the extended phi92 at a resolution of 3.9 Å. Local structures of the 5-fold region of the icosahedral head, capsid-portal, portal-adaptor, and stopper-tail of extended phi92 were reconstructed to the resolutions of 3 Å, 4.5 Å, 3.3 Å, and 3.5 Å, respectively, by combining the symmetry-mismatch reconstruction [32,33] and the local reconstruction methods [34,35] (S1C Fig). The baseplate structure of the extended phi92 was resolved to a resolution of 3.2 Å using RELION software [36] (S1C Fig). Local resolution maps for the extended phi92 are shown in S2 Fig. The high-resolution density map allowed us to build atomic models for the following extended phi92 components (Figs 1A and S3): major capsid protein (MCP, gp124), cement protein (CP, gp123), portal protein (gp120), adaptor protein (gp126), stopper protein (gp128), tail terminator protein (gp129), tail sheath protein (gp130), tail tube protein (gp131), tube initiator protein (gp135), sheath initiator protein (gp139), C-terminal region of the tape measure protein (TMP, gp134), hub protein (gp137), spike protein (gp138), plug protein (gp136), and heterotrimeric wedge complex (gp145-gp146). The N-terminus of TMP gp134 and three sets of tail fibers were not resolved to near-atomic resolution, likely due to their flexibility. The structural resolution was estimated using the Fourier shell correlation criterion with a cut-off of 0.143 according to the “gold standard” method [37].

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Fig 1. Overall structures of myophage phi92 with its extended and contracted states.

(A) Side view of the intact structure of extended phi92. (B) Cut-open view of the density map of the baseplate in the extended phi92. (C) Side view of the intact structure of contracted phi92. (D) Bottom view of the density map of the baseplate in the contracted phi92. (E) Organization of phi92 genome segments. The color code is applied to panels A-D.

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In the extended state, the icosahedral head of phi92 is composed of 775 copies of the MCP gp124 with a triangulation number T = 13. The 780 copies of the CP gp123 form 260 trimers attached to the icosahedral three-fold and quasi-three-fold axes on the head surface (Fig 1A). The head-to-tail connector (Fig 1A), anchored at a unique five-fold vertex of the head by a dodecameric portal (gp120), is composed of a dodecameric adaptor (gp126), a hexameric stopper (gp128), and a hexameric tail terminator (gp129). The tail consists of 24 hexameric stacked rings of tube (gp131) and sheath (gp130) (Fig 1A, 1B). The baseplate contains seven protein components, including a trimeric hub gp137, a trimeric spike gp138, a trimeric TMP gp134, a hexameric tube initiator gp135, a six-fold symmetric sheath initiator gp139, six heterotrimeric wedges gp145-gp146, and a six-fold symmetric plug gp136 (Fig 1B). Three sets of tail fibers are attached to the wedge of the baseplate (Fig 1A).

The same methods were used to reconstruct the structures of the head, portal-adaptor, stopper-terminator, tail sheath, and baseplate of the contracted phi92 to resolutions of 3.6 Å, 4.4 Å, 4.9 Å, 3.7 Å, and 4.2 Å, respectively (S1C Fig). Local resolution maps of the contracted phi92 are shown in S4 Fig. The contracted connector was resolved only at medium resolution, likely due to destabilization during centrifugation. Urea induces the detachment of the baseplate tip during tail contraction, resulting in the absence of detectable density (Fig 1C). Additionally, the contracted baseplate periphery remains anchored to the tail sheath and retains six-fold symmetry (Fig 1C, 1D). Using the atomic models of the extended phi92 as references, we built models for the head (gp123, gp124), the portal-connector (gp120, gp126, gp128, gp129), the tail (gp130), and the baseplate (gp139, gp145, gp146) (S5 Fig). Compared with extended phi92, contracted phi92 undergoes dramatic structural rearrangements that facilitate DNA and TMP ejection, baseplate and tail sheath contraction toward the connector, and exposure of ~460 Å of the rigid tail tube (Fig 1A, 1C). Gene products are listed in Fig 1E. Data collection and reconstruction statistics are detailed in S1 Table.

Structures of the head, portal, connector, and tail in the extended state

Based on the density map of the phi92 head, we built the atomic models of MCP gp124 and CP gp123 (Figs 2A-2C and S3B). The MCP gp124 adopts a canonical HK97 fold [38] and can be divided into four domains: N-arm, E-loop, P-domain, and A-domain. The phi92 head comprises 775 copies of the MCP gp124 organized into 11 pentons and 120 hexons (S6A Fig), forming an icosahedral shell with a triangulation number of 13. The trimeric gp123 CP adopts a β-tulip fold located at the quasi-three-fold and three-fold axes of the icosahedral head (Fig 2A, 2C), structurally similar to the CPs of phages T1 [39], lambda [40], and TW1 [41]. The CP gp123 forms tight interactions with the MCP gp124 at both the icosahedral three-fold and quasi-three-fold symmetry axes (S6B Fig). Specifically, the N‑terminus of gp123 protrudes outward to form a four-stranded β-sheet with the E-loop of an underlying gp124 and the N-terminus of a neighboring gp124 from the same capsomer, further enhancing capsid stability (S6B Fig).

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Fig 2. Structures of the head, portal-connector complex, and tail of extended phi92.

(A) Density map of an asymmetric unit of the icosahedral head corresponding to the dashed area in Fig 1A. One penton monomer and two hexons are colored yellow and light pink, respectively; the cement proteins are colored light blue. (B) Ribbon model of the MCP gp124 shown in four domains. (C) Ribbon model of the trimeric CP gp123. (D) Side view of portal-connector complex (density map). Color codes are identical to that used in Fig 1A. The insets show the zoomed-in views of the interactions between the portal and adaptor (top), and between the adaptor and stopper (bottom). (E) Ribbon model of the portal protein gp120, adaptor protein gp126, stopper protein gp128, and terminator protein gp129, colored according to their domains. (F) Side (left) and cut-open (right) views of the density maps of the tail (only terminator, three sheath and four tube rings). The color coding is identical to that used in Fig 1A. The insets show the zoomed-in views of the four-stranded β-sheet interactions between the terminator and sheath (top) and between two adjacent sheath rings (middle), and of the cation-π interaction (Y97-R373), hydrogen bond (S95-Q374) and salt bridge (E99-R373) between the tube and sheath (bottom). (G) Ribbon models of the tail tube protein gp131 and sheath protein gp130, colored according to their domains.

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The portal, occupying a unique five-fold vertex of the head, contains 12 copies of protein gp120 (Fig 2D) and exhibits high structural similarity to portals from other tailed phages [42]. Each gp120 can be divided into four domains: the crown domain, the wing domain, the stem domain, and the clip domain (Fig 2E). The clip domain provides an attachment site for the connector and interacts with the terminase motor during DNA packaging, as in phages P22 [43] and HK97 [44].

The connector (Figs 1A and 2D), attached below the portal, comprises three components: a dodecameric adaptor, a hexameric stopper, and a hexameric terminator, and exhibits structural conservation across the majority of siphophages [45–49] and myophages [20,23,50]. The adaptor protein gp126 consists of an α-helix domain, a β-sheet domain, and a C-arm (Fig 2E). The C-terminus of each adaptor gp126 extends upward and interacts closely with the outer surface of the clip domain of the portal protein, forming an augmented four-stranded β-sheet (Fig 2D). The hexameric stopper interacts with the 24-stranded β-barrel situated at the bottom of the adaptor, thereby facilitating the transition from 12-fold to 6-fold symmetry (Fig 2D). Each stopper protein gp128 adopts a typical β-barrel fold (Fig 2E). The tail terminator contains six copies of the protein gp129, each with an N-terminal domain and a β-hairpin domain (Fig 2D, 2E).

Phi92 possesses a contractile tail with a height of 960 Å (Figs 1A and 2F), composed of 24 stacked hexameric rings of tail tube protein gp131 (inner) and sheath protein gp130 (outer). The tail tube protein gp131, structurally conserved among siphophages [51,52], myophages [18,53], and CISs [54,55], consists of a β-sandwich domain flanked by an α-helix and an extended β-hairpin (Fig 2G). The tail sheath protein gp130 can be divided into an N-terminal arm, domain I, domain II, domain III, and a C-terminal arm (Fig 2G), and exhibits a high structural similarity to the sheath proteins of other myophages [15,23,56] and CISs [54,55]. Domain I of the last sheath ring interacts with the C-terminal β-hairpin domain of terminator protein gp129 via an interlaced four-stranded β-sheet (Fig 2F). Within a sheath ring, the N-arm of one gp130 and the C-arm of its neighbor, in conjunction with a two-stranded β-sheet from the domain I of a monomer in the adjacent ring, form a four-stranded β-sheet handshake interaction that bridges and stabilizes neighboring sheath rings (Fig 2F). The interactions between the first sheath ring and the sheath initiator protein are described below. In addition, each tail tube protein monomer interacts with its neighboring sheath monomer through the cation-π interaction, hydrogen bond, and salt bridge (Fig 2F).

Structure of the baseplate in the extended phi92

The baseplate of phi92 shares a conserved structural architecture with other myophages [15,20,21,23] and CISs [54,55]. It can be divided into central and peripheral regions (Fig 3A, 3B). The central region contains a hexameric tube initiator (gp135), a trimeric hub (gp137), a trimeric spike (gp138), and the C-terminus of the trimeric TMP (gp134). The peripheral region consists of six heterotrimeric wedges (gp145-gp146), a six-fold sheath initiator (gp139), a six-fold plug (gp136), and six copies each of fiber I (gp143), fiber II, and fiber III (gp147) (Fig 3B, 3C).

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Fig 3. Structures of the baseplate in extended phi92.

(A, B) Cut-open views of the density map of the central and peripheral regions. The color coding is identical to that used in Fig 1A. The peripheral region in panel A and the central regions in panel B are shown in transparency, respectively. (C) Side (left) and bottom (right) views of both the ribbon models (surface representation) of the baseplate wedges and the density maps of the tail fibers. (D, E) Ribbon models of the tube initiator protein gp135 and hub protein gp137, colored according to their domains. (F, G) Ribbon models of trimeric spike protein gp138 (F) and the trimeric C-terminus of TMP gp134 (G). The inset shows the zoomed-in view of the density map of the Fe ion (green dot). (H, I) Ribbon models of the sheath initiator protein gp139 (H) and plug protein gp136 (I). (J) Ribbon models of gp145-i (orange), gp145-o (gray) and gp146 (cyan) in a heterotrimeric wedge.

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In the central region of the baseplate, the tube initiator protein gp135 contains a tube-like domain and a flexible C-terminal loop (Fig 3D), initiating the assembly of tail tube. Hub protein gp137 comprises a tube-like domain and a C-terminal domain (Fig 3E). The tube-like domains of gp135 and gp137 exhibit folds similar to tube protein gp131, forming six-fold and pseudo-six-fold symmetric tube rings (S7 Fig), as seen in Mu [15], phiTE [17] and Pam3 [20]. Spike protein gp138 contains an oligosaccharide-binding (OB-fold) domain, a β‑helical domain, and an apex domain (Fig 3F), structurally similar to that of phage P2 [27]. Notably, the apex domain of the trimeric gp138 coordinates a ferric ion via three H × H double-histidine motifs (Fig 3F), stabilizing the tip structure for piercing the host cell membrane during infection. Our cryo‑EM structure of gp138 is consistent with the reported crystal structure [27]. In addition, the C-terminal domain (residues 631–656) of the trimeric TMP gp134 was resolved, which is situated in the baseplate interior (Fig 3A, 3G).

In the peripheral region, the six-fold sheath initiator sits at the top of the wedge, and each sheath initiator protein gp139 shares structural homology with the I domain of the tail sheath protein gp130 (Figs 3H and S8A). Similar to the inter-ring handshake mechanism, gp139 forms a four-stranded β-sheet handshake interface with the N-arm of one sheath protein and the C-arm of a neighbor in the first sheath ring (S8B Fig), firmly anchoring the baseplate to the sheath. The sheath initiator protein is critical for sheath assembly and contraction, and is highly conserved across CISs [57,58] and other myophages [19,22]. Six copies of plug protein gp136 are arranged around the hub periphery, serving as a docking site for the wedge (Fig 3B). Each plug protein gp136 adopts an Ig-like fold structure (Fig 3I), a conserved structural and functional feature among myophages. The wedge is composed of six heterotrimers assembling into a ring-like structure (Fig 3C). Each heterotrimer contains one gp145 monomer and two conformational variants of the gp146 monomer, referred to as gp146-i located at “inner” portion of the wedge ring and gp146-o located at “outer” portion of the wedge ring (Fig 3J). The heterotrimer adopts a radial orientation: gp145 and gp146-o face the baseplate periphery (Fig 3J), while the pin domain of gp146-i faces the baseplate center, forming part of the inner interface adjacent to the central spike and hub (Fig 3B). The N-terminal segment of each monomer assembles into a core helical bundle, while their C-terminal domains adopt a canonical trifurcated fold (Fig 3J).

Structures of the three fibers in the extended phi92

The phi92 phage assembles three distinct types of tail fibers, each comprising six trimers attached to the baseplate wedge (Fig 3C). Fiber I adopts an L-shaped conformation and connects to the upper end of the C-terminal domain of wedge protein gp145 (Fig 3C). Fiber II connects to the base of fiber I and extends downward at ~30° (Figs 3C and S3A). Fiber III connects to the bottom of the C-terminal domain of gp145 and points downward at ~37° (Figs 3C and S3A). In comparison with resolution previously reported [9,26], local reconstruction of fiber III using cryoSPARC improved its resolution to approximately 7 Å (S9A Fig). Although insufficient for atomic modeling, the N-terminal domain of the AlphaFold3-predicted gp147 trimer fits well with the density map, supporting the identification of fiber III as gp147 (S9B–S9C Fig). The predicted gp147 trimer exhibits structural similarity to the trimeric fibers of phages Mu [15] and Milano [18] (S9D Fig). Attempts to resolve fibers I and II using the same approach did not improve resolution; however, we provided detailed hypotheses regarding their structures and functions.

Capsular polysaccharides on the bacterial surface resist phage infection by acting as a physical barrier. Phage phi92 must penetrate a thick polysaccharide capsule to infect strains like E.coli K1 and K92 [12,24], indicating that capsule degradation is a crucial step for phi92 to initiate infection. These capsules consist mainly of polysialic acid with α2,8- or α2,9-linkages [59,60]. Biochemical experiments have confirmed that phi92 encodes endosialidase gp143, a bifunctional enzyme hydrolyzing both types of polysialic acid [12], explaining its broad host range. Although gp143 density was not discernible in our cryo-EM map, sequence analysis reveals that gp143 shares 51%, 52%, and 54% identity with endosialidases of phages K1E [61], K1-5 [62], and K1F [63], respectively (S10A Fig). AlphaFold3 trimeric predictions of endosialidases of phages K1E, K1-5 and K1F (S10A Fig) and the crystal structure of K1F endosialidase [64] reveal a characteristic mushroom-shaped conformation and a putative active site, similar to the gp143 structure in phi92, suggesting that these proteins share a common evolutionary origin and functional conservation. To date, only low-resolution tail fiber structures of K1E and K1-5 have been reported [65]. Their fibers share an L-shape architecture, with an arm-like scaffold protein binding an endosialidase at the distal end. The density of fiber I of phi92 is conformationally similar to the tail fibers of K1E and K1-5 (S10B Fig). The predicted trimeric structure of phi92 gp143 fits the density corresponding to the catalytic domain at the fiber tip (S10C Fig). Based on these comparisons, we speculate that the distal region of fiber I corresponds to the trimeric gp143 of phi92.

Although the extremely low resolution of the fiber II density in the extended baseplate precluded atomic model building, fiber II plays a pivotal role in the initial infection stages of phage phi92. Therefore, we employed AlphaFold3 to predict the structures of all proteins encoded by the phi92 genome and systematically screened them for candidates capable of adopting tail fiber-like architectures. Based on structural topology comparisons with tail fibers or spikes from other phages [66,67], we identified four putative tail fiber proteins, gp15, gp140, gp150, and gp151, with the exception of gp143 and gp147 (S11 Fig). In particular, the previous report [25] indicates gp150’s potential role in degrading the colanic acid layer on the bacterial surface, thereby facilitating the phage in breaching the exopolysaccharide barrier. We performed a structural homology search of gp15, gp140, and gp151 using the Dali server [68]. The results revealed that gp15 lacks homologous structures; Gp140 and gp151 share high homology with the head spike protein gp52 of phage Pa223 [69], but the function of these proteins remains uncharacterized. However, the AlphaFold3-predicted structures of these candidate proteins did not fit well into the low-resolution density map of fiber II. Notably, these structures also showed poor fitting to the previously reported density map of the corresponding fiber in the extended state of phi92 [9], where gp150 was speculated to be fiber II. Currently, the structural and genomic prediction of fiber II remains a significant challenge.

Structural changes of the head-tail complex in the extended and contracted states

Urea treatment triggers conformational transition from the extended to contracted state, accompanied by significant changes in the baseplate and tail sheath, leading to TMP and genomic DNA release (Fig 4A) and yielding a contracted phi92 particle with an empty head, a contracted sheath, and an intact baseplate (Figs 1B and S5A).

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Fig 4. Structural changes of the portal-connector-tail complex of phi92 in the extended and contracted states.

(A) Cut-open views of the density maps of the portal-connector-tail complex in extended phi92 (left) and urea-treated contracted phi92 (right). The color coding is identical to that used in Fig 1A. (B) Superimposition of the sheath monomers in the extended and contracted states showing the conformation changes. (C) Top views of the conformational changes of the tail from the extended to the contracted states. (D) Ribbon models of all sheath monomers in one helical pitch of extended (light blue) and contracted (dark green) states along tail tube (pink).

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In both states, the structures of the head, the portal, and the head-to-tail connector remain nearly unchanged, as observed in most siphophages [70] and myophages [15,19,23]. Notably, density for the C-terminal β-hairpin of the terminator protein gp129 was not resolved in the contracted state (Fig 4A). Based on terminator conformational changes in other myophages like E217 [23], Mu [15], and XM1 [19], we hypothesize that the flexible C-terminal β-hairpin domain of gp129 undergoes similar conformational rearrangement to maintain connection with the tail sheath during contraction. Structural analysis shows tube protein gp131 remains largely unchanged, while sheath protein gp130 undergoes substantial tertiary and quaternary rearrangements. At the tertiary structure level, structural superposition shows the N‑arm rotates ~100°, the C‑arm shifts ~8 Å, and domain I rotates ~12° (Fig 4B), indicating each sheath monomer undergoes rigid‑body rotation mediated by these regions during contraction. At the quaternary structure level, all sheath subunits remain connected via the four-stranded β-sheet handshake interaction (Figs 2F and S12), but the diameter of the sheath ring increases from 136 Å to 160 Å, disrupting the sheath-tube interface (Fig 4C). Helical twist and rise of the sheath change from 26.32° and 38.63 Å (extended) to 32.74° and 17.34 Å (contracted) (Fig 4D), resulting in a tighter sheath packing and exposure of ~460 Å of the rigid tail tube (Fig 1C). This contraction mechanism is conserved in myophages E217 [23] and Mu [15], and CISs like R-type pyocins [55] and PVCs [54]. Specifically, contraction transitions the tail sheath from a high-energy extended state to a low-energy contracted state, and the released energy drives tail tube penetration into the host cell membrane for genome delivery [13,71]. This contraction propagates wave-like along the tail axis toward the connector [72–74].

Structural changes of the baseplate in the extended and contracted states

Urea-induced tail contraction in myophage phi92 causes the dissociation of the baseplate peripheral region from the central region (Fig 1C). High-resolution structure of the contracted baseplate central region was not obtained, so its protein composition remains unconfirmed. Density maps of phi92 baseplate show plug and spike dissociate from the tail tip (Fig 1C, 1D), consistent with phage E217 [23], suggesting conserved functions: the plug acts as a wedge docking site and may disengage upon wedge conformational change; the spike likely mediates the host outer membrane penetration and then dissociates to create a conduit for the TMP and genome release.

Structural change in the baseplate periphery during tail contraction is critical for stabilizing the fully contracted conformation of the tail apparatus. Structural comparisons between the extended and contracted states (Fig 1A, 1C) show that fiber I position remains essentially unchanged, while fiber III reorients from a downward-tilted conformation (extended) to a downward orientation perpendicular to the tail axis (contracted). A similar conformational change occurs in T4 short fiber during T4 contraction [22], suggesting fiber III rearrangement may transmit the contraction signal to the wedge. Fiber II was not observed in the contracted baseplate. Similarly, in phage A511, partial density of one type of its fibers was missing due to the urea-induced tail contraction [73]. Therefore, it is possible that the urea treatment during the preparation of the contracted sample severely disrupted the local structure of fiber II or its binding stability with the baseplate, causing it to detach from the tail and resulting in the subsequent loss of its signal. Morphologically, fiber II appears short, thick, and rigid, with structural features that likely share infection dynamics similar to the reported P22 gp9 [75] and Sf6 gp14 [76,77]. Structures of this type undergo subtle conformational changes and exhibit no obvious swinging movements during infection.

Comparison of the heterotrimeric wedge structures in the extended and contracted states reveals that the core helical bundle remains largely unchanged, maintaining trimeric unit stability. However, the C-terminal domains of gp146-i and gp146-o undergo significant conformational changes, rotating approximately 36° clockwise and 15° counterclockwise, respectively (Figs 3J and 5A). In the extended baseplate, the C-terminal domains of gp146-i and gp146-o from adjacent heterotrimeric wedges form a dimerization interface via a handshaking interaction (S13A Fig). In the contracted baseplate, they move apart, transitioning the dimerization interface into a fingertip-like interaction (S13A Fig). These changes expand the wedge ring diameter from 56 Å to 108 Å (Fig 5B), accompanied by tail tip release. Although wedge rearrangements are conserved across CISs [57,78] and myophages [22,23], specific conformation details vary.

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Fig 5. Structural changes of the baseplate in extended and contracted states.

(A) Superimposition of a heterotriplex complex in the extended and contracted states. (B) Top view of the conformational changes from the extended wedge (right) to the contracted wedge (left). (C) Superimposition (top) and structural comparison (bottom) of the sheath initiator protein in the extended and contracted states. (D) Top view of the conformational change of the sheath initiator ring from extended (right) to contracted (left) states. All monomers in the rings are colored magenta, except for one monomer (colored red) from the extended ring and one monomer (colored blue) from the contracted ring.

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Sheath initiator protein gp139, though low in molecular weight, is crucial for structural integrity between the baseplate periphery and contractile tail sheath (S13B Fig). Comparison shows its C-terminal arm undergoes significant conformational change during contraction (Fig 5C), cooperatively facilitating subunit expansion and rotation. Notably, the gp139 C-terminus interacts closely with the baseplate wedge (S13C Fig). Thus, wedge ring expansion drives the expansion of the hexameric sheath initiator ring, increasing its diameter from 88 Å to 120 Å (Fig 5D). In addition, protein gp139 maintains strong interaction with the first sheath ring in the contracted state, forming a reinforced four-stranded β-sheet handshake structure (S12D Fig). The inclination angle of the handshake structure increases from 27° (extended) to 48° (contracted) (Fig 5C). These changes in gp139 trigger structural rearrangements in the first sheath layer, propagating wave-like upward through successive layers, ultimately driving global sheath contraction.