In situ structures of the portal-neck-tail complex of bacteriophage T4 inform a viral genome positioning mechanism

Domain: chemistry
Published: 2026-02-20
Editorial outcome: minor_revision

Abstract
The post-genome packaging mechanisms that govern the assembly of an infectious virion are poorly understood in bacteriophages and other viruses. Here, our near-atomic resolution cryo-EM structural analyses uncovered an assembly-and conformation-driven genome positioning mechanism in the tailed bacteriophage T4. We show that following headful packaging, which generates a pressurized head, a global conformational change occurs in the portal structure, probably triggering packaging termination and ejection of the packaging motor. Our high-resolution structures of the neck of the virion further show that the neck undergoes conformational changes upon docking of a pre-assembled tail onto the sealed neck, which then opens a genome-gate. Driven by the pressure of the packaged DNA, the genome travels through open neck channels, binds and compresses the resident tape-measure protein, and halts at the bottom of the second topmost disk of the tail tube. Pressure-suspended within the virion’s innermost tunnel and secured by a baseplate plug, the genome remains poised to flow through the tunnel into a host cell upon receiving the host receptor recognition signal.

Introduction
After encapsidating the genome into a proteinaceous capsid to near crystalline density, icosahedral bacteriophages (phages) and other viruses must safely deliver the entire genetic material into a new host cell. Loss of even a small segment of the genome could be detrimental and result in a noninfectious virion. We recently discovered a genome retention mechanism in the tailed myophage T4 that seals off the pressurized capsid with a “neck” assembly attached to the unique dodecameric portal vertex2. Our high-resolution structures revealed a closed “genome-gate” formed by two sets of “stoppers”, one from the hexameric neck protein gp14, and another from a nucleic acid-binding E. coli host protein Hfq, which is also a hexamer2.

A pre-assembled tail would then bind to this neck connector followed by attachment of long tail fibers to the baseplate to complete the infectious virion assembly3–6. What, if anything, happens to the genome during this process and up until delivery into a new host cell remains unknown. It was generally believed that the genome seal might remain in place until delivery, perhaps reinforced by the attachment of the tail to the neck. The seal would then be broken upon receiving a signal from a baseplate conformational change and tail sheath contraction upon host receptor recognition by the tail fibers7–9. To investigate this mechanism, we have determined the in situ cryo-electron microscopy (cryo-EM) structures of the infectious phage T4 virion.

Here, we describe the high-resolution asymmetric reconstruction of the portal-neck-tail connector complex and reconstructions of specific segments of the phage tail. The cryo-EM maps allowed near-atomic resolution of many of the virion components in situ. These include: the dodecameric portal protein gp20; the neck proteins gp13 (dodecamer) and gp14 (hexamer); the hexameric tail terminator protein gp15; the hexameric tube terminator protein gp3; the tail terminator-tail sheath (gp18) junction, the tube terminator-tail tube (gp19) junction; segments of the tape-measure protein (TMP) gp29; and the TMP-baseplate hub protein (gp27) junction. Importantly, besides the atomic descriptions, these reconstructions revealed a series of structural and conformational transitions that led to an entirely unanticipated genome positioning mechanism.

First, the in situ structures showed a global structural transition in the dodecameric portal assembly of the DNA-full mature virion when compared to the empty tail-less capsid10 or the in vitro-assembled portal-neck complexes. The portal dodecamer changed from its flying saucer-shaped structure in the empty capsid to a mushroom-shaped structure in the DNA-full capsid of the mature virion. Moreover, the entire portal dodecamer moves “down” by ~10 Å with respect to the capsid shell, presumably as a result of the ~25-35 atm internal pressure due to tight “headful” packing of the highly negatively charged genome11–17. This transition most probably triggers termination of packaging, ejection of packaging motor, and exposure of binding sites for the neck assembly.

Second, major conformational transitions occur in the neck assembly. A domain in gp13 swings upwards, embracing the portal, while the gp14 loops that form the genome-gate swing downwards, interacting with the tail. Strikingly, unlike in the in vitro-assembled portal-neck intermediate, where the neck channel is closed by a double genome-gate, in the mature virion, the channel is completely open. Moreover, the genome end descended further down into the tail tube, and the tape-measure protein (TMP) gp29 is compressed. Our reconstructions show extended coiled-coil segments of TMP as a narrow “tube” within the ~40 Å-diameter gp19 tail tube. The TMP spans the tail tube all the way down to the baseplate hub where it is bound by the pseudo-six-fold-symmetric gp27 trimer of the cell-puncturing device, the gp27-gp5-gp5.4 complex.

These structural analyses lead to a conformation-driven genome positioning mechanism in which the pressurized genome in the finished virion is no longer restrained by the neck assembly’s genome-gate. Attachment of the pre-assembled tail to the neck triggers dramatic conformational changes in the neck structure, causing expulsion of the Hfq stopper and opening of the gp14 genome-gate. The DNA pressure then pushes the genome through the neck-gp15-gp3 connector channels, where it is captured by the resident TMP’s N-terminal DNA-binding tip. The DNA-TMP complex is then further pushed down by the pressure into the gp19 tail tube, compressing the TMP tube. The genome thus is pressure-suspended in the innermost tunnel of the finished virion, extending from the head into the portal, the neck, and the tail tube, showing no discernible interactions with any of the components of this long tunnel. In total, the DNA travels ~170 Å after tail attachment, starting from the genome-gate down to the second topmost disk of the tail tube, where it remains poised to flow into a new host cell upon receiving the host cell receptor recognition signal.

Results
Cryo-EM of the phage T4 virion
Wild-type (WT) phage T4 virions twice-purified by CsCl gradient centrifugation were frozen onto cryo-EM grids and examined with a Titan Krios electron microscope equipped with a K3 Summit detector (Supplementary Fig. 1a). Cryo-EM data were collected comprising 12,176 micrograph movies, and three-dimensional reconstructions focused on different parts of the T4 virion were calculated (Table 1 and Supplementary Fig. 2). These include the asymmetric and 6-fold symmetric reconstructions focused on the phage neck, which have resolutions of 3.8 Å and 3.3 Å, respectively. The reconstructions included the portal, the neck proteins, the tail terminator, and the respective interfaces at the top region of the tail. In addition, we report the 3-fold and 6-fold-symmetric reconstructions of the middle part of the tail, which had resolutions of 3.4 Å and 3.3 Å, respectively; and 3-fold and 6-fold-symmetric reconstructions focused on the phage baseplate with resolutions of 3.6 Å and 3.4 Å, respectively. The maps showed continuous density for the polypeptide chains and recognizable side-chain densities (Supplementary Fig. 1). The atomic structures were built and refined against the cryo-EM maps (Table 1). The structures were then combined to generate the whole portal-neck-tail complex (Supplementary Fig. 2d).

A global structural transition in the portal assembly
Our in situ asymmetric structure of the portal assembly in T4 virion consists of twelve molecules of gp20 (525 residues; 61 kDa) arranged in a mushroom-like structure with a central channel (Fig. 1). Each subunit consists of the four classic domains found in our previously reported T4 portal structures10,18 and in many other phage/viral portals19: the clip domain, the stem domain, the wing domain, and the crown domain (Fig. 1a).

The portal’s central channel has a diameter of ~40 Å in which resides a single thread of DNA double helical genome that has a diameter of ~20 Å. The DNA is freely suspended in the channel, showing no significant interactions with the portal lumen (Fig. 1b and Supplementary Fig. 3) including notably where the tunnel loops (residues 378-396) project into the channel. This suggests that the portal channel morphed into an “open” state in the infectious virion. Significantly, previous genetic and biochemical studies11,19–22 indicate that the tunnel loops bind to the last packaged DNA and stabilize it while awaiting neck assembly (see below).

The shape of the portal dodecamer in the DNA-full virion is remarkably different from that in the empty neck/tail-less capsid (Fig. 1c–h)10, or the in vitro-assembled portal-neck complexes2. While the DNA-empty portal structure has the shape of a flying saucer, the DNA-full virion portal has a mushroom-like shape (Fig. 1c, d). Moreover, in the DNA-full virion, the center of mass of the portal dodecamer is pushed down by ~10 Å, and the clip domain by ~15 Å with respect to the capsid shell, presumably by the pressure of the highly condensed genome to near crystalline density (~500 mg/ml)11,12,14,23. These differences reflect a global conformational transition involving a major structural reorganization of the portal (Supplementary Movies 1–3; Fig. 1c–h).

Morphing between the portal structures from the empty and full heads shows concerted movements of portal’s structural elements that reposition and/or remodel the domains (Supplementary Movie 3). The crown and clip domains change their relative orientations with respect to the wing and stem domains (Supplementary Movie 3, Fig. 1g, h). Consequently, the channel diameter widens at the clip region from ~30 Å to ~40 Å, while it constricts at the crown region from ~44 Å to ~38 Å (Supplementary Movies 2, 3). The portal tunnel loops, which are flexible and exposed into the channel constricting its diameter in the empty heads, assume a rigid and flattened conformation in the full heads through interactions with residues from the stem and crown domains, causing the channel to be completely open. The stem helices forming the channel change angles from ~55° to ~40° relative to the central axis, while the clip domain is remodeled exposing new binding sites for the neck protein gp13 (Supplementary Movie 3).

Most probably, this structural transition is induced by the tightly packed acidic DNA (“headful packaging”) that creates ~25-35 atm pressure inside the capsid11,13,17,24, which then pushes on the wing and crown domains, causing the portal’s displacement relative to the capsid shell. Consequently, the DNA around the portal is stabilized, showing well-resolved rings of density in the cryo-EM map (Supplementary Fig. 3). These DNA rings would be in steric clashes with the portal structure observed in the empty capsid, which is also consistent with the above model that headful packaging, probably near the end of the process, induces the portal conformational change. This portal transition then transmits the headful packaging signal from the capsid interior to the clip domain at the capsid exterior, resulting in ejection of the packaging motor, and exposure of binding sites for assembly of the neck protein gp13. The neck assembly then may cause additional conformational changes in the portal and the neck to further stabilize the structure (see below).

The above analyses demonstrate a switch in the portal conformation from a symmetry-mismatched motor-assembling platform to a symmetry-matched neck-assembling platform.

Conformational transitions in the neck assembly
Gp13 dodecamer. The 309-aa phage T4 gp13 neck protein binds to the portal protein at 1:1 ratio after the packaging motor’s departure (Fig. 2a–d). Each gp13 subunit in the virion structure comprises three domains and an extended C-terminal region (Fig. 2e and Supplementary Fig. 4), all well-resolved and exhibiting distinct interactions with different components; portal, head, and Wac (whisker antigen control) fibers (Fig. 2, Supplementary Figs. 5, 6). The four-helix bundle domain I consisting of the two first and the two last α-helices of the gp13 chain (Fig. 2e and Supplementary Fig. 4) forms the core, having a fold similar to much smaller, usually single-domain ortholog proteins from other phages, such as SPP125,26, HK9727, XM128 and P2229. Twelve domains I form a dodecameric ring that extends the portal tunnel (Fig. 2a, d) and its inner wall is lined by the C-terminal negatively charged α-helices (residues 280-300). This core gp13 dodecamer, the structure and interactions of the gp14-binding loops emanating from the core (residues 266–279 and 17–31), and the C-terminal portal-binding extension (residues 301-309) which inserts between the clip domain helices (Supplementary Fig. 5a), are all similar in both the virion and the in vitro-assembled neck complexes.

On the other hand, the “swing” domain II (residues 54-188) shows remarkable conformational changes in the virion when compared to the in vitro-assembled neck intermediates (Fig. 2e, f; Supplementary Movie 4; Supplementary Fig. 6). Most notable is the very different orientation this domain adopts in the two structures (Supplementary Fig. 6c, d; Supplementary Movie 4). While it forms the peripheral ring of the dodecamer with no interactions with the portal protein in the in vitro-assembled structure (Supplementary Fig. 6), in the virion the domain swings upwards by ~90° (Supplementary Movie 4), with one side of the β-sandwich formed by two β-sheets interacting with the portal’s clip domain α-helix 295-309 and the hairpin 311-325 (Supplementary Fig. 5b). Furthermore, the long flexible and partly α-helical “anchor” region 90-157 (Fig. 2e, g) inserts into the gap between the portal and the capsid shell and interacts with the stem and wing domains of the portal and the P (periphery) domains of the gp23* major capsid protein (Fig. 2f). Notably, the side chains of cysteines 125 of gp13 are located in the proximity of cysteines 245 from the portal’s wing domains potentially forming an S-S bonds (Supplementary Fig. 5c). Because of the symmetry mismatch between the gp13 dodecamer and five-fold-symmetric capsid shell, the flexible anchor regions of the gp13 subunits adopt different conformations (Supplementary Fig. 4b) adjusting to different capsid environments. However, the rest of the gp13 polypeptide chain has a similar conformation in all gp13 subunits and obeys the strict twelve-fold symmetry of the dodecamer.

The T4 gp13 also has a special domain III (residues 190-239) not found in other phages (Fig. 2e and Supplementary Fig. 7), which is not resolved in the in vitro-assembled neck structure due to its flexibility, but in the virion, it models into a binding site for interaction with the N-terminal domains of fibritin molecules, trimers of gpWac. Twelve fibritin molecules attached to gp13 dodecamer decorate the virion’s neck, with six fibritins forming a propeller-shaped collar and the other six fibritins oriented downwards as whiskers30.

Gp14 hexamer. The 256-residue neck protein gp14 assembles on the dodecameric gp13 as a hexamer (Fig. 3a–e). Gp14 completes the neck assembly and serves as an attachment point for the independently assembled phage tail30,31. As was predicted previously30, the core fold of the gp14 neck protein has some similarity with analogous neck proteins from other phages, as well as the tail tube proteins, such as T4 gp19.

While the basic networks of interactions between the gp14 hexamer and the gp13 dodecamer are nearly the same in the finished virion and the in vitro-assembled neck intermediate, the virion gp14 structure shows key conformational changes that are linked to attachment of the tail (Supplementary Movies 5, 6, Supplementary Fig. 8).

First, most remarkable, the ~40 Å diameter central channel of gp14 ring is completely open (Fig. 3c, f) in the virion while housing the double-helical DNA. The stopper loops (residues 94–112) that close the genome-gate in the in vitro-assembled intermediate (Supplementary Fig. 8a) undergo a ~90° rotation downward. Furthermore, parts of these loops (residues 100–111) interact with the hexameric tail terminator protein gp15 in the assembled virion (Fig. 3g). Second, unlike in the in vitro-assembled intermediate neck structure, the gp14 hexamer is no longer bound to the Hfq protein. Third, in the full virion, the gp14 structure shows an extended negatively charged C-terminal region (212-245) containing an α-helix and a “hook” segment (residues 231-245), which extensively interacts with the hexameric tail terminator protein, gp15 (Fig. 3d–h; see below). On the other hand, in the in vitro-assembled intermediate, this region is disordered and not resolved. These changes reflect key conformational transitions in gp14 (Supplementary Movies 5, 6) when it encounters gp15 present at the top region of the ~20 MDa pre-assembled tail, leading to Hfq ejection, opening of the genome-gate, and movement of DNA down into the tail structure (see below).

Structure of the tail terminator protein gp15 and the neck-tail interface
During tail assembly, a hexameric ring of gp15 (Fig. 4a–c) attaches to the top of the tail and terminates the tail growth4, hence the name “tail terminator”. Like gp14, gp15 has fold similarity to the phage tube proteins30. Although the core in situ cryo-EM structure of the gp15 ring is similar to our previously reported crystal structure of the recombinantly expressed gp1530, there are important conformational changes and interactions with gp14 and the gp18 sheath.

Each gp15 molecule contains two loop regions 28-74 and 176-184, located at the top of the gp15 ring (Fig. 4d) that extensively interact with the gp14 regions 65-76 and 100-111 (part of the stopper loop) located at the bottom of the gp14 hexamer (Fig. 3g). Additionally, the helix and hook segments of the gp14 C-terminal region embrace the gp15 ring forming five salt-bridges between negatively charged D212, E220, D222, E227 and E233 of gp14, and positively charged R74, K46, R38, R11 and K160 of gp15 (Fig. 3d). These interactions repeated six times between the gp14 and gp15 hexamers generate a massive ~27,000 Å2 interface and a strong neck-tail connection.

Phage T4 gp15 has an unusual 64-residue C-terminal domain (residues 209-272) which is essential for interactions with the tail sheath and probably is the key to terminate the tail growth (Fig. 4). This domain, containing a β-sheet formed with three anti-parallel β-strands, was not resolved in the previously reported crystal structure probably because of its flexibility in the absence of the sheath interactions. Each C-terminal domain from the gp15 hexamer attaches to the C-terminal domain of a gp18 subunit from the topmost ring of the contractile sheath.

Furthermore, our cryo-EM map allowed building of an atomic model for the entire gp18 protein, including its C-terminal domain (Fig. 4e). In the previously resolved truncated mutant structure (gp18M)32, this key C-terminal domain was missing. This domain (residues 548-631) contains two anti-parallel β-strands and two α-helices, which are also conserved in the sheath proteins of other myoviridae phages and contractile injection systems5,33.

The β-strands from both the gp15 and gp18 C-terminal domains interact, forming a shared five-stranded β-sheet (Fig. 4e). In addition to reinforcing the tail structure34, these interactions likely terminate sheath polymerization during the tail assembly by capping the last gp18 ring. Additionally, later in the infection mechanism, these would also stabilize the contracted sheath-neck connection when the sheath detaches from the tail tube.

Of note is that the tail terminator proteins of other myoviridae phages also have similar C-terminal segments which interact with the sheath, suggesting a common sheath termination mechanism28,35–37. However, in other phage orthologs, the C-terminal regions are much smaller with fewer interactions when compared to T4 gp15. For example, the phage XM1 tail terminator protein has a C-terminal region containing only 13 residues and one β-strand which interacts with the sheath and augments a β-sheet in the C-terminal domain of its sheath protein28. Thus, it appears that, during evolution, T4 gp15 acquired a larger C-terminal domain to generate a stronger, more structurally reinforced neck-sheath connector.

Structure of the tube terminator protein gp3 and its interfaces with the gp19 tube, the gp15 tail terminator, and the gp18 sheath
Phage T4 has a special hexameric tube terminator protein, gp3, which attaches to the top of the tube during the tail assembly and terminates the polymerization of the tail tube protein gp1934. In the assembled tail, the gp3 ring is sandwiched between the gp19 tube (at the bottom) and the gp15 hexamer (on top) (Fig. 5a–c). The gp3 structure is very similar to that of the gp19 tube protein despite a low sequence identity, ~22%. The core of a gp3 subunit consists of a β-sandwich flanked by an α-helix (Fig. 5d). The hexameric ring of gp3 contains flexible loops on its top and bottom surfaces, which interact with gp15 and gp19, respectively (Fig. 5d–f). Each gp3 subunit interacts with three subunits of gp15. Namely, the loop region 145-150 of gp3 interacts with two gp15 subunits, whereas the loop region 117-121 and the elongated N-terminal segment of gp3 (residues 2-15) extensively interact with a third gp15 subunit (Fig. 5e).

At the bottom of the ring, gp3 contains a long loop region (residues 45–65), which attaches to three subunits of the gp19 tube, and a second loop region (residues 97–105), which interact with two other gp19 subunits. Thus, each gp3 subunit interacts with five gp19 chains creating strong binding networks with the tube (Fig. 5f).

Additionally, the gp3 hexamer interacts with the C-terminal domains of gp18 from the topmost ring of the contractile sheath. Specifically, the β-sheet forming the outer surface of the gp3 hexamer interacts with the α-helix from the gp18 C-terminal domain, stabilizing the sheath (Fig. 5g). Such an intricate network of interactions between gp3, gp19, gp15, and gp18 terminates the polymerization of the tail tube as well as generates a stable pre-assembled tail structure for attachment to the neck.

The tape-measure protein anchors the terminal region of the genome inside the tail tube
It has been well-documented through previous genetic studies that gp29 is a tape-measure protein (TMP), acting as a ruler that determines the tail length4,38–42. During the tail assembly, one end of TMP attaches to the baseplate hub and helps the polymerization of the tube protein gp19, which assembles into a stack of hexameric rings around TMP. The tube growth continues until the end of the ruler is reached, where the hexameric tube terminator protein gp3 attaches to the last ring of the tube (and also probably to the ruler), terminating gp19 polymerization. Therefore, in the pre-assembled tail structure, the tip of the TMP ruler is expected to be located near the end (tip) of the tube and gp3, an architecture well-supported by genetic and structural studies in several myoviridae and siphoviridae phages6,43–45. Surprisingly, however, our cryo-EM maps show that in the mature T4 phage virion, the TMP is no longer associated with the tip of the tube. Instead, the upper end of the TMP is now associated with the end of the genomic DNA and has moved down into the tube.

The maps show a continuous rod-like density inside the portal-neck-tail connector region beginning at the portal’s crown, then extending down to the bottom of the second topmost ring of the gp19 tube (~320 Å) (Fig. 6). We attribute this density to the terminal segment of genomic DNA, showing no interaction with the portal or neck proteins. Clearly, the portal and neck channels are open, which is consistent with the conformational changes observed in the neck causing the genome-gate to open.

Moving down into the tail structure, reconstructions focused on the middle part of the tail and the baseplate show a stretch of density inside the gp19 tail tube which spans from the baseplate to the DNA terminus (Fig. 6a). This density is attributed to the tape-measure protein (TMP, gp29). At the top is the N-terminus of TMP in association with the DNA, the DNA-TMP junction (Fig. 6b), based on the genetic evidence46, while the C-terminus is at the bottom. The 20-residue C-terminal region of TMP is well-resolved in our 3-fold-symmetric reconstruction focused on the baseplate. The cryo-EM map shows three C-terminal regions of TMP interacting with the trimeric (pseudo-hexameric) baseplate hub protein, gp27, located at the center of the baseplate (Supplementary Fig. 9). These baseplate hub-TMP interactions may form an initiation complex that lead to the initiation of the tail assembly4.

In the middle of the tube, our cryo-EM reconstructions show two tube-like density segments (~150 Å and ~200 Å-long) which, most probably, correspond to coiled-coil regions of the TMP. These segments showed six-fold-symmetric features even when the reconstruction symmetry was released to C3. Models composed of hexameric coiled-coils show reasonable agreement with the cryo-EM densities (Supplementary Fig. 10), and are also consistent with a similar hexameric coiled-coil structure observed in a genome delivery intermediate we have recently discovered46 and the direct mass determination by scanning electron microscopy47.

Together, these cryo-EM reconstructions suggest that the TMP gets compressed by the descending DNA when the genome-gate opens. In total, the pressurized DNA travels ~170 Å distance, first by passing through the open neck connector (gp14, gp15, and gp3) channels and then by compressing the TMP into the tail tube. This means that the tail attachment involves not mere docking of the tail tip on the sealed head, but by dynamic conformational transitions that pressure-suspend the DNA-TMP complex in the innermost tunnel of the virion in a metastable state. The genome, thus, remains poised for rapid and smooth release into a new host cell during infection46.

Methods
Cryo-EM sample preparation and data collection
WT T4 phage particles were prepared from E. coli infected lysates using standard protocols52,53. To produce particles with high purity, the phage was subjected to two CsCl gradient centrifugations. The final phage preparation was flash-frozen and stored in PBS at −80 °C.

The cryo-EM grids were prepared using the Vitrobot Mark IV (Thermo Fisher Scientific) operated at 4 °C with 100% humidity. Aliquots (3.5 μl) of the purified phage sample were applied onto ultra-thin lacey carbon 400 mesh grids (Ted Pella catalog No: 01824) that had been glow-discharged using a PELCO easiGlow system for 60 sec at 25 mA. Excess sample was blotted away after a 15 sec wait time using a blotting force of 2 and a blotting time of 5 sec. The grids were plunge-frozen in liquid ethane and stored in liquid nitrogen until data collection.

Cryo-EM data were collected at the Purdue University Cryo-EM Facility using a 300 kV Titan Krios G4 cryo-electron microscope equipped with a Gatan BioQuantum K3 detector (Thermo Fisher Scientific). A total of 12,176 micrograph movies (each composed of 60 frames) were recorded using the EPU software with a nominal magnification of 42k, corresponding to a pixel size of 1.026 Å, and a defocus range of −0.6 to −2.0 μm. The total electron dose was 38 e/Å2. The motion correction and calculation of the dose weighted micrographs were performed using CryoSPARC54. The CTF parameters were estimated using the ctffind4 program55.

3D reconstructions
Cryo-EM reconstructions were calculated using the Relion software package56,57. First, 5390 phage capsids were manually selected from the micrographs and the capsid particles (1280 pixels) were extracted and subjected to 2D classification. The class averages were used for the Relion automatic picking procedure. A total of 540,975 boxes, produced by auto-picking, were subjected to 2D classification, and 195,670 capsid particles, belonging to good classes, were selected for calculation of the 3D capsid reconstruction. To speed up calculations, the particles were rescaled to the pixel size of 3.648 Å (360 pixels). The 3D reconstruction was calculated using the Relion auto-refine procedure, using the previously reported cryo-EM structure of the capsid58 filtered to 40 Å resolution as an initial reference map. The D5 symmetry was applied during the reconstruction process. The reconstruction had a resolution of 7.3 Å.

Then the relion_particle_symmetry_expand program was used to expand each orientation entry in the data file generated by capsid reconstruction procedure into ten D5 related entries in a new STAR file. The new file was used to extract particles moving the box centers 175 pixels (638.4 Å) along the Z-axis of the map (from the center of the capsid to the neck). Initially 256 × 256 boxes with the pixel size of 1.603 Å were extracted, and an initial reference was generated using the relion_reconstruct program using the particle’s orientations obtained in the previous reconstruction. The extracted boxes were subjected to 3D classification imposing the C6 symmetry without alignment, and 164453 good neck-centered particles were selected. For those particles, larger boxes of 400 × 400 pixels with the pixel size of 1.616 Å were extracted. The 3D reconstruction was performed using the Relion auto-refine procedure imposing the C6 symmetry. The map had a resolution of 3.3 Å based on the “gold-standard” Fourier shell correlation (FSC) criterion using the 0.143 cutoff59. This map included the portal vertex of the capsid with surrounding capsomers, the neck proteins and the top part of the tail. In this map, the major capsid protein and the anchor region of gp13 were washed out because they do not follow the imposed symmetry.

Then the box centers were moved 472 Å along the Z-axis, from the neck to the middle region of the tail, and the new 400 × 400 boxes with the pixel size of 1.616 Å were extracted. Particles that had the centers outside the micrographs were removed. An initial reference was generated by the relion_reconstruct program using the particles’ orientation obtained in the previous (neck reconstruction) step. A total of 157,789 particles were used to calculate the 3D reconstruction of the middle part of the tail using the Relion auto-refine procedure. The C6 symmetry was imposed during the reconstruction process and the particles’ orientations were locally refined. The final map had a resolution of 3.3 Å.

Subsequently, the box centers were further moved 540 Å along the Z-axis, from the middle region of the tail to the baseplate, and new 400 × 400 boxes with the pixel size of 1.616 Å were extracted. Particles that had the centers outside the micrographs were discarded. An initial reference was generated by the relion_reconstruct program using the particles’ orientation obtained in the previous reconstruction. A total of 151,499 particles were then used to produce the reconstruction of the baseplate region using the Relion auto-refine procedure. The C6 symmetry was imposed during the reconstruction and the particles’ orientations were locally refined. The final map had a resolution of 3.4 Å.

Next, the C3-symmetric reconstruction of the baseplate was calculated. For that, the relion_particle_symmetry_expand was used to generate a particle stack expanded with C6 symmetry. The particles were subjected to the masked 3D classification without alignment. The mask covered only the cell-puncturing device gp5-gp27, which has the 3-fold symmetry. A total of 113,841 particles were selected after the classification. The relion_reconstruct program was used to generate a new reference map. The reconstruction was then calculated using the Relion auto-refine procedure. The particles’ orientations were locally refined, and the C3 symmetry was imposed during the reconstruction process. The final map had a resolution of 3.6 Å.

Subsequently, the box centers were moved back to the middle part of the tail, by −540 Å along the Z-axis to calculate the 3-fold symmetric reconstruction of the middle part of the tail. The relion_reconstruct program was used to generate a reference map. The reconstruction was then calculated using the Relion auto-refine procedure. The particles’ orientations were locally refined, and the C3 symmetry was imposed during the reconstruction process. The final map had a resolution of 3.4 Å.

Finally, the asymmetric (C1) reconstruction centered on the phage neck region was calculated. The relion_particle_symmetry_expand program was used to expand each orientation entry in the data file generated by the 6-fold symmetric neck reconstruction procedure into six C6-related entries in a new STAR file. The particles were subjected to the masked 3D classification without alignment using the C5 symmetry. The mask covered the major capsid protein capsomers (the 5-fold-symmetric part of the particle), and the previous neck-centered reconstruction was used as an initial reference map. A total of 69,107 particles were selected after the classification. The relion_reconstruct program was used to generate a new reference map. Then the asymmetric reconstruction was calculated using the Relion auto-refine procedure. The final map had a resolution of 3.8 Å.

The cryo-EM maps colored by local resolution are shown in Supplementary Fig. 11, and the Fourier shell correlation curves for the reconstructions are shown in Supplementary Fig. 12. The workflows for the reconstructions are presented in Supplementary Figs. 13 and 14. The angular distribution of the particles used in the reconstructions are shown in Supplementary Fig. 15.

Model building and refinement
The atomic structures were built using Coot60. To build the portal protein and surrounding major capsid protein capsomers, the previously reported structures from the empty T4 capsid were used as a starting model10. The gp13 and gp14 neck protein structures were built de novo. For the gp15 tail terminator, the previously reported crystal structure30 was used as a starting model. The gp3 tube terminator structure was built de novo. The C-terminal domain of the sheath protein gp18 was built de novo. For the rest of the gp18 structure, the crystal structure of the gp18 C mutant was used as a starting model32. For the fibritin N-terminal regions, the crystal structure of the N-terminal domain61 was used as a model. For the tail tube and the baseplate, the previously reported cryo-EM structure of the baseplate-tube complex was used as a model8. The atomic structures were refined in real space using Phenix62,63 against the asymmetric reconstruction centered on the neck and the 3-fold symmetric reconstructions of the baseplate and the middle part of the tail. The refinement statistics are summarized in Table 1.

The overlapping regions of the reconstructions were aligned, and the reconstructions were combined using ChimeraX64,65 to generate the map of the entire portal-neck-tail complex. The atomic structures were fitted into the combined map to create the combined model. The figures and movies were generated using ChimeraX64,65.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This paper ”In situ atomic structures of the portal-neck-tail complex of bacteriophage T4 inform a viral genome positioning mechanism” by Fokine at al presents a comprehensive analysis of phage T4 genome packaging, which is enhanced by clear and easy-to-follow figures. The authors make two significant discoveries in T4 biology that greatly advance current understanding of this model system. First, the finding that the portal protein ‘sinks’ into the capsid after genome ejection is intriguing. Second, the well-defined structure of the tape measure protein is highly relevant, particularly in relation to genome ejection. The accompanying videos are very helpful and greatly clarify a complex assembly process. Overall, this paper demonstrates the depth and quality of research expected for publication in *Nature Communications*.\n\nMain comments\n1 - Figures 3 and 4 are less critical and could be moved to the Supplementary Information. Similar structures are found in many other bacteriophages. The authors could save space here and focus on the tape measure protein and baseplate (see next comments).\n2 - Figure 6 shows top and bottom views, along with enlarged views of the ordered region of the tape measure protein. How clear is the electron density? Add an overlay of the model and the density map. Also, the area with weaker density might indicate a disordered part of the protein lacking an α-helical structure. How does the AlphaFold prediction appear for this protein?\n3 – The cryo-EM map’s resolution appears lower in the baseplate compared to the tail/neck. The authors should fit the structure solved by crystallography into the cryo-EM map and model this key structural element. The paper otherwise feels incomplete.\n4 – The data collection and refinement table should be part of the main text since these structures are new. Also, I was not able to locate FSC curves in the Supplemental Figures. In the Table, please indicate the resolution of all cryo-EM maps at FSC = 0.5 and 0.143.", "reviewer_2": "The cryo-EM structure of mature bacteriophage T4 described in this paper provides important insights into an intermediate state of virus assembly following the attachment of the viral tail. It shows, for the first time, that tail assembly in T4, and likely in other related bacteriophages, triggers the opening of the DNA gate within the connector and induces a substantial (17 nm) translocation of DNA inside the connector/tail tunnel, bringing it into direct contact with the tape measure protein. Comparison of this structure with those of earlier intermediates in virus assembly reveals substantial conformational changes in several proteins that prevent DNA leakage at different stages of virus assembly and, upon opening of the DNA gates, enable its release during infection, providing significant insights into understanding the mechanism of virus maturation and host infection. The paper is clearly written, excellently illustrated, and will be of broad interest to the virology and structural biology communities.\n\nMain points that need addressing:\nAbstract.\nText after the second sentence (“Here, our near-atomic…”) should be revised to clearly distinguish the structural findings reported in this paper from any speculations or proposed mechanisms. Furthermore, the third and fourth sentences (“Following headful packaging…” and “Opening the genome gate…”) appear to describe findings from other studies (e.g., Han et al., submitted?) rather than results from the present work. These should either be explicitly attributed to appropriate publications or rephrased to avoid confusing these findings with the current paper’s contributions.\nL154 “This portal transition then transmits the headful packaging signal from the capsid interior to the clip domain at the capsid exterior resulting in remodeling of the clip domains, ejection of the packaging motor, and exposure of binding sites for assembly of the neck protein gp13. “ - I would argue that it is equally plausible that exposure of the clip domain leads to its interactions with the neck protein, and conformational changes in the clip are induced by this interaction, and not by headful pressure.\nL342. “Previous genetic and biochemical studies indicate that the portal channel is probably closed transiently during this event and the DNA is restrained by the tunnel loops. Deletion of parts of the tunnel loops resulted in leakage of the packaged genome generating noninfectious virus particles19-21. “ - It has been previously demonstrated that the portal protein can transit between closed and open conformations, working as a one-way valve to prevent DNA leakage after motor detachment (doi: 10.7554/eLife.55517) - could a similar mechanism be employed by other bacteriophages, including T4? It is plausible that subsequent interactions with gp13&neck complex factors induce conformational changes that reopen the portal tunnel.\nLine 713. Extended Figure 6(a) - Authors reference “Han et al., submitted.” Is this the correct paper or did the authors intend to reference Sun et al., 2025 (doi: 10.21203/rs.3.rs-6853951/v1)? Can the authors include a full citation if the paper has been published as a preprint?\nMinor points:\nL69: “The portal dodecamer changed from its flying saucer-shaped structure in the empty capsid to a mushroom-shaped structure “ and L132 “While the DNA-empty portal structure has the shape of a flying saucer, the DNA-full virion portal has a mushroom-like shape“ - I am not convinced such comparison is appropriate as the overall shape of the portal doesn’t change as much, its internal tunnel remains well-defined, with significant conformational changes observed only within individual domains along with some inter-domain adjustments.\nL96: “…where it remains poised to flow smoothly and rapidly…” - it would be worth de-dramatising this sentence, as it is not known whether the movement is in fact “rapid” nor has the speed of such movement been established.\nL251-253: “The β-strands from both the gp15 and gp18 C-terminal domains interact...these interactions likely terminate sheath polymerization during the tail assembly.” - Is there a reference that supports this statement?", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We thank the reviewer for the positive comments and a highly enthusiastic summary of our work.\nMain comments\n1 - Figures 3 and 4 are less critical and could be moved to the Supplementary Information. Similar structures are found in many other bacteriophages. The authors could save space here and focus on the tape measure protein and baseplate (see next comments).\nFigures 3 and 4 show the hexamers of the stopper protein, gp14, and the tail terminator protein gp15, which form the head-tail interface. These proteins have unique features compared with orthologs from other phages. Namely, gp14 has an extended C-terminal region containing the helix and ‘hook’ segments which embrace the gp15 hexamer through the formation of 30 salt-bridges that reinforce the head-tail attachment. The gp15 protein has an unusual 60-residue C-terminal domain extensively interacting with the topmost ring of the gp18 sheath, strengthening the tail structure.\nFurthermore, these figures show an “open” genome gate with the DNA passing through both the gp14 and gp15 hexamer channels, an important aspect of the genome positioning mechanism.\nTherefore, it is important to show these distinctive structural features in the main figures, which also flows smoothly with the text.\n2 - Figure 6 shows top and bottom views, along with enlarged views of the ordered region of the tape measure protein. How clear is the electron density? Add an overlay of the model and the density map. Also, the area with weaker density might indicate a disordered part of the protein lacking an α-helical structure. How does the AlphaFold prediction appear for this protein?\nWe thank the reviewer for these comments and addressed them as follows:\nOur reconstruction focused on the baseplate allowed us to resolve the 20-residure C-terminal region of the tape measure protein (TMP), gp29. The density is clear and shows the side chains, as shown in the Supplementary Figure 1d. We have modified the figure legend to make these points clearer.\nThe interactions of the gp29 C-terminal region with gp27 that is part of the cell-puncturing device (gp27-gp5-gp5.4 complex) are also shown in Supplementary Figure 9c. We have enlarged this figure in the revised manuscript.\nThe gp29-gp27 interactions might be important for the formation of a tail assembly initiator complex made of the hub and TMP proteins. We stated this point on page 15.\nOur reconstruction of the middle part of the tail shows tubular regions of gp29 density located inside the gp19 tail tube. We interpret these regions as ordered coiled-coil segments, although the density is not fully resolved. As the reviewer points out, weaker density for other parts of gp29 may indicate disordered regions. Alternatively, a weaker density may correspond to an ordered structure which has somewhat different orientations/positions in different phage particles. Thus, averaging between different particles would produce weaker density. Per the reviewer’s suggestion, we have added a figure showing an overlay of the coiled-coils and the density in the revised manuscript (Supplementary Figure 10).\nSecondary structure prediction programs (Jpred, PSIPRED) suggest that gp29 has mostly alpha-helical structure. We attempted to model the full-length gp29 using AlphaFold, however it did not generate an extended gp29 structure. Gp29 evidently adopts its extended conformation only when the gp19 tail tube assembles around it. In solution, gp29 likely exists in a flexible, non-extended form. Therefore, it is not surprising that AlphaFold did not produce an extended gp29 model.\n3 – The cryo-EM map’s resolution appears lower in the baseplate compared to the tail/neck. The authors should fit the structure solved by crystallography into the cryo-EM map and model this key structural element. The paper otherwise feels incomplete.\nWe fitted the previously determined baseplate structure (Taylor et al., 2016, doi: 10.1038/nature17971) into our 3.6 Å-resolution C3-symmetric reconstruction focused on the baseplate. We adjusted the atomic structure using Coot and refined the structure in real space using Phenix (Table 1). The structure was deposited to PDB and will be released with this paper. The lower resolution of the baseplate reconstruction can be explained by the structural flexibility of the baseplate. The baseplate contains six wedges surrounding the central hub, which are less rigid compared with the middle part of the tail and the neck.\nUnlike in the previously published reconstructions, our reconstruction here allowed us to resolve the baseplate hub and the C-terminal region of gp29 attached to it (Supplementary Figures 9 and 1d).\nIn this paper, we have not included detailed descriptions of the baseplate structure in order to focus on the genome positioning mechanism, and also because the baseplate proteins were already described in Taylor et al., 2016, doi: 10.1038/nature17971 and also in our previous study (Yap et al., 2016, doi: 10.1073/pnas.1601654113), which we cited in the paper.\n4 – The data collection and refinement table should be part of the main text since these structures are new. Also, I was not able to locate FSC curves in the Supplemental Figures. In the Table, please indicate the resolution of all cryo-EM maps at FSC = 0.5 and 0.143.\nWe thank the reviewer for these comments. As suggested, we have moved the data collection and refinement table to the main text. As the reviewer suggested, the resolution is now shown for all maps at FSC = 0.143 (gold standard) and at FSC = 0.5. The FCS curves were in the validations reports which we uploaded. Now we have also added a new Supplementary Figure 12 in the revised manuscript showing all FSC curves in the revised manuscript.\nWe thank the reviewer for the positive comments and a highly enthusiastic summary of our work.\nMain points that need addressing:\nAbstract.\nText after the second sentence (“Here, our near-atomic…”) should be revised to clearly distinguish the structural findings reported in this paper from any speculations or proposed mechanisms. Furthermore, the third and fourth sentences (“Following headful packaging…” and “Opening the genome gate…”) appear to describe findings from other studies (e.g., Han et al., submitted?) rather than results from the present work. These should either be explicitly attributed to appropriate publications or rephrased to avoid confusing these findings with the current paper’s contributions.\nWe have modified the Abstract accordingly.\nL154 “This portal transition then transmits the headful packaging signal from the capsid interior to the clip domain at the capsid exterior resulting in remodeling of the clip domains, ejection of the packaging motor, and exposure of binding sites for assembly of the neck protein gp13. “ - I would argue that it is equally plausible that exposure of the clip domain leads to its interactions with the neck protein, and conformational changes in the clip are induced by this interaction, and not by headful pressure.\nWe thank the reviewer for this comment. In fact, we have considered this model suggested by the reviewer. One concern was that, in this model, the motor had to be first ejected to expose the clip domains without a conformational change. If so, in this (motor-interacting) conformation, the clip domains will not have as high affinity to interact with the gp13 neck proteins. This means that it risks DNA leakage until the clip domains change conformation and bind the gp13 neck proteins to seal the packaged head.\nTherefore, it is more plausible that a headful signal triggers the portal conformational change causing ejection of the motor since the clip domains no longer retain binding affinity for the motor, while allowing the pre-assembled gp13 neck complex to lock into the portal since the exposed clip domains have changed to gp13-binding conformation. This would allow efficient neck assembly and seal the packaged head.\nAdditionally, our reconstruction shows rings of DNA around the portal protein in the fully assembled virion (Supplementary Fig. 3). These DNA rings would be in clashes with the portal structure observed in the empty capsids, suggesting that when the capsid becomes full, the portal does not stay in its original “empty” conformation and undergoes conformational changes, probably near the end of packaging and before neck attachment. However, our model does not exclude further conformational changes induced by the neck attachment.\nWe have modified the text on page 8 to include the above points.\nL342. “Previous genetic and biochemical studies indicate that the portal channel is probably closed transiently during this event and the DNA is restrained by the tunnel loops. Deletion of parts of the tunnel loops resulted in leakage of the packaged genome generating noninfectious virus particles19-21. “ - It has been previously demonstrated that the portal protein can transit between closed and open conformations, working as a one-way valve to prevent DNA leakage after motor detachment (doi: 10.7554/eLife.55517) - could a similar mechanism be employed by other bacteriophages, including T4? It is plausible that subsequent interactions with gp13&neck complex factors induce conformational changes that reopen the portal tunnel.\nIt is possible that the T4 portal acts as a one-way valve by allowing DNA packaging while preventing DNA leakage. However, we don’t have enough evidence, since the tunnel loops were not resolved in our previous portal reconstructions from the empty capsids. The reconstruction of the assembled virion, reported here, does resolve the tunnel loops and shows that the tunnel is open (ready for DNA ejection).\nWe however agree that it is possible that neck attachment may induce conformational changes in the portal, opening the channel. We have modified the text on page 16 to include this point and also cited Bayfield et al., 2020 paper (https://doi.org/10.7554/eLife.55517) in the revised manuscript.\nLine 713. Extended Figure 6(a) - Authors reference “Han et al., submitted.” Is this the correct paper or did the authors intend to reference Sun et al., 2025 (doi: 10.21203/rs.3.rs-6853951/v1)? Can the authors include a full citation if the paper has been published as a preprint?\nAs suggested by the reviewer, we have included the citation of this preprint.\nMinor points:\nL69: “The portal dodecamer changed from its flying saucer-shaped structure in the empty capsid to a mushroom-shaped structure “ and L132 “While the DNA-empty portal structure has the shape of a flying saucer, the DNA-full virion portal has a mushroom-like shape“ - I am not convinced such comparison is appropriate as the overall shape of the portal doesn’t change as much, its internal tunnel remains well-defined, with significant conformational changes observed only within individual domains along with some inter-domain adjustments.\nWe appreciate this comment. However, this descriptive analogy is helpful to easily remember and visualize the conformational change. Moreover, the portal dodecamer does change shape significantly with the domains moving relative to each other as shown in the Videos S1 and S3.\nL96: “…where it remains poised to flow smoothly and rapidly…” - it would be worth de-dramatising this sentence, as it is not known whether the movement is in fact “rapid” nor has the speed of such movement been established.\nAs the reviewer suggested, we removed “smoothly and rapidly”.\nL251-253: “The β-strands from both the gp15 and gp18 C-terminal domains interact...these interactions likely terminate sheath polymerization during the tail assembly.” - Is there a reference that supports this statement?\nThe C-terminal domain of gp15 attaches to the top of the last hexametric ring of the gp18 sheath. Thus, gp15 attachment would prevent binding of any additional sheath subunits. Additionally, Zhao et al., (2003) showed that gp15 stabilizes the sheath structure. We have modified the corresponding sentence on page 12. Now it reads: “In addition to reinforcing the tail structure (Zhao et al., 2003), these interactions likely terminate sheath polymerization during the tail assembly by capping the last gp18 ring.”\nThank you for co-reviewing our paper!"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have adequately addressed my comments and further enhanced the paper. As shown in the cryo-EM table and the revised Supplementary Figure 12, the resolution of all cryo-EM maps is impressive, and the atomic models are well refined. I commend them for adding a new Supplemental Figure 10 that displays the hexameric regions of TMP inside the tail tube. Overall, I believe this paper is ready for publication in Nature Communications."}, "author_response": ""}]

Decision letter