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# About bacteriophage tail terminator and tail completion proteins: structure of the proximal extremity of siphophage T5 tail

Romain Linares, Cécile Breyton

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Romain Linares, Cécile Breyton. About bacteriophage tail terminator and tail completion proteins: structure of the proximal extremity of siphophage T5 tail. Journal of Virology, 2025, 99, pp.e0137624. 10.1128/jvi.01376-24. hal-04931720

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# About bacteriophage tail terminator and tail completion proteins: structure of the proximal extremity of siphophage T5 tail

Romain Linares,¹ Cécile Breyton¹

**AUTHOR AFFILIATION** See affiliation list on p. 11.

**ABSTRACT** Bacteriophages are viruses infecting bacteria. The vast majority of them bear a tail, allowing host recognition, cell wall perforation, and DNA injection into the host cytoplasm. Using electron cryo-microscopy (cryo-EM) and single particle analysis, we determined the organization of the tail proximal extremity of siphophage T5 that possesses a long flexible tail and solved the structure of its tail terminator protein p142 (TrP₁₄₂). It allowed us to confirm the common evolutionary origin between T5 TrPp142 and other known or putative TrPs from siphophages, myophages, and bacterial tail-like machines, despite very poor sequence conservation. By also determining the structure of the T5 tail proximal extremity after interaction with T5 bacterial receptor FhuA, we showed that no conformational changes occur in TrPp142 and confirmed that the infection signal transduction is not carried by the tube itself. We also investigated the location of T5 Neck1 or tail completion protein p143 (TCPp143) and showed, thanks to a combination of cryo-EM and structure prediction using Alphafold2, that it is not located at the capsid-to-tail interface as suggested by its position in the genome, but instead, very unexpectedly, on the side of T5 tail tip, and that it appears to be monomeric. Based on structure comparison with other putative TCPs predicted structures, this feature could not be shared by other TCPs and questions the affiliation of p143 to this family of protein.

**IMPORTANCE** Bacteriophages, viruses infecting bacteria, are the most abundant living entities on Earth. They are present in all ecosystems where bacteria develop and are instrumental in the regulation, diversity, evolution, and pathogeny of microbial populations. Moreover, with the increasing number of pathogenic strains resistant to antibiotics, virulent phages are considered a serious alternative or complement to classical treatments. 96% of all phages present a tail that allows host recognition and safe channeling of the DNA to the host cytoplasm. We present the atomic model of the proximal extremity of the siphophage T5 tail, confirming structural similarities with other phages. This structure, combined with results previously published and further explored, also allowed a review and a discussion on the role and localization of a mysterious tail protein, the tail completion protein, which is known to be present in the phage tails, but that was never identified in a phage structure.

**KEYWORDS** bacteriophage T5, cryo-electron microscopy, tail terminator protein, tail completion protein

Bacteriophages—phages for short—are viruses infecting bacteria. They represent the most abundant biological entity on our planet. A large majority of them are composed of a capsid filled with double-stranded DNA and a tail, which can be long and contractile (myophage), long and flexible (siphophage), or short (podophage). The distal extremity of the tail bares the host-recognition apparatus, which also allows the

**Editor** Kristin N. Parent, Michigan State University, East Lansing, Michigan, USA

Address correspondence to Cécile Breyton, cecile.breyton@ibs.fr.

The authors declare no conflict of interest.

See the funding table on p. 12.

**Received** 6 August 2024

**Accepted** 15 November 2024

**Published** 23 December 2024

Copyright © 2024 Linares and Breyton. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
perforation of the bacterium cell wall and the safe channeling of the DNA into the host cytoplasm. In long-tailed phages, the assembly pathway of the capsid and the tail are independent, and DNA-filled capsids attach onto functional tails to form fully assembled virions at the end of the lytic cycle. In siphophages, functional tails are produced after capping of the tail tube by the tail terminator proteins (TrP), of which siphophage λ gpU is the prototype (1). In myophages, tails can be capped both by a tube terminator and by a sheath terminator, as in T4 (2), or only by a tail terminator, which caps both the tube and the sheath, as in P1 (3) or Pam3 (4). In some phages, the tail needs the addition of the tail completion protein (TCP) to form fully functional tails, of which λ gpZ is the prototype (5). In some publications, both names are used interchangeably to designate the tail terminator. Empirically, it has been observed that phage proteins within the tail are arranged in a similar order to their coding genes in the genome (5, 6). Thus, it is expected that the TCP, also called Ne1 (Neck protein type 1) in (6, see also https://phrogs.lmge.uca.fr/), encoded by a gene usually located downstream of the capsid module and upstream of the TrP of the tail module, itself upstream of the tail tube protein, would also be located at the capsid-to-tail interface, the neck (5–7).

Phage T5 that infects *E. coli* belongs to the morphotype siphophage, which accounts for the majority of tailed phages. In the structure module of its genome, under the control of the same promoter and located between the head completion protein gene and the tail tube protein gene, are found two genes, p142 and p143 that were attributed to the TrP and the TCP, respectively (Fig. 1A and H) (8): $TrP_{p142}$ was indeed localized within the virion in the neck using cross-linking with anti-TrP$_{p142}$ IgG and immuno-gold labeling combined with EM imaging (8). A similar approach for TCP$_{p143}$ proved unsuccessful: TCP$_{p143}$ could be overexpressed in some conditions, but the protein precipitated on the Ni-NTA column, preventing the obtention of purified protein to raise antibodies (9). We recently determined the structure of the T5 tail tip—the distal extremity of the tail—before and after interaction with its receptor (10), where we proposed that a monomer of TCP$_{p143}$ be attached to the Baseplate Hub Protein pb3 (BHP$_{pb3}$). Here, we present the cryo-EM structure of phage T5 tail proximal extremity at a pseudo-atomic resolution, also before and after interaction with the phage receptor. $TrP_{p142}$ is resolved as a hexameric ring, interacting with the last tail tube protein TTP$_{pb6}$ ring. No available density could account for TCP$_{p143}$ in this region of the tail.

## RESULTS AND DISCUSSION

### Structure of the proximal extremity of phage T5 tail

T5 tails, purified from a capsid defective mutant, were produced, vitrified for cryo-EM characterization, and imaged for the data collection on a high-end electron microscope (Fig. S1A) (10). Image analysis yielded a map of T5 tail proximal extremity, containing a trimeric ring of the tail TTP$_{pb6}$, on top of which sits a hexameric ring formed by the $TrP_{p142}$ (Fig. 1E). The estimated resolution of this map is 3.9 Å (Fig. S1C and D), which allowed us to build pseudo-atomic models for both $TrP_{p142}$ hexamer and a TTP$_{pb6}$ trimer. $TrP_{p142}$ is a 161-residues protein, mainly formed by a four-strand antiparallel β-sheet that lines the lumen of the tube, flanked by two α-helices, and a short loop between β-strands 1 and 2. An 18-residue β-hairpin links the second α-helix to the β-sheet (Fig. 1F). As already discussed in the case of phage λ TrP gpU (11), $TrP_{p142}$ fold appears to be related to the canonical tail tube domain (TTD) fold, a common building block present in numerous phage proteins (12, 13), although clear differences are present (Fig. S1G). It indeed exhibits a similar β-sheet/helix/loop fold, but a second α-helix is present in $TrP_{p142}$ while a second β-sheet is present in the TTD. Also, the orientation of the helices is different (Fig. 1G). The interaction between two neighboring $TrP_{p142}$ monomers is mostly mediated through strand β1, N-terminus, and C-terminus for monomer 1, and strand β3, α-helix 2, and the 22–29 loops for monomer 2. The interaction between the hexameric $TrP_{p142}$ ring and the trimeric TTP$_{pb6}$ ring under it is ensured by the tip of loop 63–67, which inserts in two similar pockets resulting in the domain duplication of TTP$_{pb6}$ (14) and is mostly mediated by electrostatic interactions (Fig. S2).
<figure><img src="image_4.png" /><figcaption>**FIG 1** (A) Scheme of T5 tail organization. The proximal end of the tail (formed of TrP<sub>p142</sub> and TTP<sub>pb6</sub> rings) is framed in red and the upper part of the distal complex (comprising TCP<sub>p143</sub>, DTP<sub>pb9</sub>, and BHP<sub>pb3</sub>) is indicated by a black accolade. (B) Top (upper panel) and side (lower panel) views of the TrP<sub>p142</sub> hexamer as found at the distal extremity of the tail. Two consecutive monomers are not related by a C6 symmetry and are displayed in two different shades of blue. The outer and inner dimensions of the ring are indicated. (C) Superimposition of two TrP<sub>p142</sub> consecutive, non-symmetry related, monomers. (D) Superimposition of two TrP<sub>p142</sub> monomers from native tails (blue) and tails after interaction with receptor FhuA (white). (E) Side view of the proximal extremity of the tail, featuring the TrP<sub>p142</sub> hexamer (in shades of blue) sitting on top of the TTP<sub>pb6</sub> trimer (in cyan). (F) Annotated model of TrP<sub>p142</sub>, indicating its main secondary structures. C and N-termini are also indicated. (G) Superimposition of TrP<sub>p142</sub> (blue) and a tail tube domain from TTP<sub>pb6</sub> (yellow). (H) Map of T5 tail structural proteins genes.</figcaption></figure>

As the TrP<sub>p142</sub> hexameric ring sits on top of a TTP<sub>pb6</sub> trimeric ring, it exhibits a C3/
pseudo-C6 symmetry and two consecutive TrP<sub>p142</sub> monomers are not strictly identical
(Fig. 1B and C). However, TTP<sub>pb6</sub> also exhibits a C3/pseudo-C6 symmetry as it results in
domain duplication (14) (excluding its Ig-like domain); thus, the two consecutive TrP<sub>p142</sub>
monomers are almost superimposable (rmsd = 1.02 Å over 156 residues). This is reminiscent of what was observed for T5 distal tail protein DTP<sub>pb9</sub>, which also assembles as a
hexamer sitting above a trimer (10).

Interestingly, purified TrPs crystallize as biologically relevant hexamers (PDB 3FZB (1) 2GJV and 4ACV). Also, a previous study showed that over-expression of TrP<sub>p142</sub> with a His<sub>6</sub>-tag in the N-terminus leads to soluble, monomeric protein while over-expression of TrP<sub>p142</sub> with a His<sub>6</sub>-tag in the C-terminus (TrP<sub>p142-Cter</sub>) leads to the formation of fibers, of a diameter similar to that of T5 tails (9). This illustrates the fact that phage structural proteins have not only evolved to adopt a specific structure, allowing them to be building blocks for the phage but also to bear inherent dynamics when monomeric. This allows them to remain monomeric, even at high concentrations, and to polymerize and assemble *only* in presence of their partners, to form a fully functional virion. Polymerization however also occurs when conditions are extreme, as in a crystallization drop. In the case of TrP<sub>p142-Cter</sub>, the tag addition somehow modifies the dynamics of the protein, which induces aberrant polymerization upon over-expression. TrP<sub>p142-Cter</sub> fibers do not look like empty tubes, as would be expected by the piling of TrP<sub>p142</sub> rings. It could be that the C-terminal tag folds back in the lumen of the tube, filling it and explaining why affinity purification failed (9). Several structures of TTP<sub>pb6</sub> were previously solved, in
different contexts: either as a crystallized monomer, within the tail tube, or interacting with the DTP<sub>pb9</sub> (10, 14). Our study adds the structure of TTP<sub>pb6</sub> in contact with the TrP<sub>142</sub> to these known structures. Overall, there are very little variations between the different TTP<sub>pb6</sub> structures and TTP<sub>pb6</sub> in interaction with TrP<sub>142</sub> superimposes almost perfectly with TTP<sub>pb6</sub> interacting with another TTP<sub>pb6</sub> or with DTP<sub>pb9</sub> (rmsd of 0.655 Å and 0.576 Å, respectively, over the 464 residues).

DALI searches link TrP<sub>142</sub> to several phage TrPs from siphophages as well as from myophages, bacterial tail-like systems related to myo- or siphophages or prophage proteins (Fig. 2A through D; Table S1), with DALI Z-scores ranging from 23.9 to 5.4 and alignment rmsd from 1.2 to 4.2 Å, even though sequence identity is non-existent (7 to 18%). This confirms that TrPs form a family of largely conserved proteins among sipho-, myophages, and related machinery, despite no sequence homologies. Several structures of siphophage TrPs are available, either from purified protein or within the phage context: gp17 from SPP1 (15–17), gpU from λ (1, 18–20)(Fig. 3C and D), gp18 from R4C (21), and gp119 from phage DT57C (22), determined by NMR, X-ray crystallography, and/or cryo-EM. *In phago* structures confirm that TrPs indeed assemble as a hexamer, at the proximal extremity of the tail tube, thereby terminating tube polymerization and providing the surface for interaction with the capsid (17–19, 22). Deletion of this protein has various phenotypes: in phage λ, capsids are produced along with very long, TMP-less, polytails, resulting from the endless polymerization of the TTP (23); in myophages T4 and P2, deletion mutants of the tube terminators gp3 and gpR, respectively, result in

<figure><img src="image_5.png" /><figcaption>**FIG 2** Comparison of TrP<sub>142</sub> with TrPs homologous proteins using DALI pairwise. TrP<sub>142</sub> is displayed in all panels, colored in dark blue, and superimposed to a selection of homologous proteins of different categories. (A) Siphophages and siphophage-like machines, phage λ (dark green), GTA (light green). (B) Myophages, phage T4 (yellow), phage XM1 (white). (C) Phage-like machines related to myophages: PyocinR2 (red), PVC (orange). (D) Miscellaneous: Type4 fimbrial biogenesis protein PilO from *P. aeruginosa* (gray).</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**FIG 3** Isosurface view of the non-sharpened cryo-EM maps of T5 tail proximal extremity, in its native state (A, gray) and after interaction with the bacterial receptor FhuA (B, cyan). Left: side interior view and right: top view. The position and thickness of the top view slice are indicated by black accolades in the side view slice. TMP<sub>pb2</sub> is indicated with red arrows in A and is absent in B. The framed panel in A shows the upper part of the tail tube extremity at a higher threshold, with TMP<sub>pb2</sub> running up to the very end of the tube. (C) Slices of a C3 map of the proximal extremity of the T5 tail tube at different positions, showing TMP<sub>pb2</sub> inside the lumen.</figcaption></figure>

tails with a longer tube, whereas deletion mutants of the sheath terminators gp15 and gpS, respectively, bear longer sheaths (24–26); in myophages Mu and SPO1, deletions mutants of the TrP K and 8, respectively, result in longer tails. In contracted tails, the tubes protrude from the capsid-proximal extremity of the tails rather than from the base plate (27, 28). In siphophage SPP1, normal-length tails are assembled in deletion mutant of the TrP gp17, which however cannot attach to full capsids (29). Thus, TrPs are suggested to both allow to stop the tail polymerization at the right length, together with the TMP (30), but also to allow interaction with the capsid (29). Some TrPs are larger proteins and include additional domains, for example interacting with the sheath of the myophage tails when they also fill the sheath termination role. Interestingly, the PVC16 TrP C-terminus additional domain appears to share structural similarity with the TrP<sub>p142</sub> fold, indicating domain duplication, a common “LEGO” strategy among phages and phage-related machines (13).

## The tape measure protein TMP<sub>pb2</sub>

The unsharpened cryo-EM map of the T5 tail proximal extremity shows a 25 Å diameter large, cylindrical, and ill-defined density filling the tail tube lumen to the top of TrP<sub>p142</sub>, which disappears from the map after interaction with FhuA (Fig. 3A and B). This density most probably belongs to the N-terminus of the Tape Measure Protein pb2 (TMP<sub>pb2</sub>), which fills the entire tail tube in native phages and purified tails (14). Unlike for the distal part of the tail, the resolution of the central density in this proximal extremity of the tail is too low to propose a stoichiometry, yet alone a model of the TMP. However, as a trimer was solved for the C-terminus of the protein (10), it is reasonable to propose that a trimer is also present in the N-terminus. Also, and despite the low resolution, different slices taken along the cryo-EM map of the T5 tail proximal extremity do not seem to hint at a higher oligomeric state than a trimer of TMP<sub>pb2</sub> (Fig. 3C).

Indeed, TMP stoichiometry has remained uncertain for many decades. In 1973, Zweig and Cummings measured the relative abundance of the different proteins of
phage T5, based on a band densitometry analysis of radiolabeled phage proteins after SDS-PAGE (31). TMP<sub>pb2</sub>, BHP<sub>pb3</sub>, and the straight fiber protein pb4 were observed to represent 1.5%, 1.5%, and 1%, respectively, of the structural proteins of phage T5. In 1988, McCorquodale and Warner interpreted these data and concluded that TMP<sub>pb2</sub>, BHP<sub>pb3</sub>, and pb4 were present as pentamers in the virion (32). In 1974, Casjens and Hendrix proposed a stoichiometry of 6–7 per virion for TMP<sub>gpH</sub> of phage λ, based on densitometry measurements of phage protein bands after SDS-PAGE and Coomassie blue or Fast green staining (33). Both in T5 (10, 22) and λ (18, 19), however, a trimer of the TMP was solved in the C-terminus of the protein, in tight interaction with the BHP, as for siphophages 80α (34). For T5, this is consistent with Zweig and Cummings’ results, as BHP<sub>pb3</sub> and pb4 were also found to be trimers (10). A stoichiometry of six has been however proposed for the TMP of myophages Pam3 (4) and A-1(L)(35). More data, at higher resolution, is thus needed to clarify this point.

TMP<sub>pb2</sub> reaches the proximal extremity of the tube (Fig. 3A), indicating a possible direct interaction with the DNA in the full phage, as seen in other siphophages (e.g., SPP1 (15), λ (18)). As previously proposed (10, 14), the expulsion of the TMP would be part of the signal transmission pathway that starts with host recognition by the receptor binding protein, continues with cell wall perforation, and ends with DNA ejection. All along the tube and except for the 35 last residues in the tail tip, TMP<sub>pb2</sub> does not interact with the tube wall (10, 14), which can explain the low resolution observed for that protein; this is the case in all phage tail structures determined to date.

## Structure of the proximal extremity of the tail after interaction with receptor FhuA

We also imaged T5 tails after interaction with *E. coli* receptor FhuA reconstituted into nanodiscs (Tail-FhuA) with cryo-EM (10). Image analysis yielded a second 3D map for the proximal extremity of Tail-FhuA, at an average resolution of 4 Å (Fig. S1C and D) allowing us to build pseudo-atomic models for TrP<sub>p142</sub> and TTP<sub>pb6</sub>. At the resolution of our maps, no conformational change was observed in TrP<sub>p142</sub> monomers or hexamer (rmsd 0.43 Å over 156 residues between Tail and Tail-FhuA individual monomers) (Fig. 1D), and neither in TTP<sub>pb6</sub>. For the latter, it was already shown that the T5 tail tube, formed of a stack of TTP<sub>pb6</sub> trimeric rings, does not undergo any conformational changes upon interaction with FhuA and that the signal of the infection is not transmitted from the receptor to the capsid through the tube itself (14). TrPs homologous proteins from myophages or tail-like machines also show no conformational change upon infection (2, 36, 37).

Because TMP<sub>pb2</sub> is expelled during the infection process (8, 10), the lumen of the tube is empty in Tail-FhuA (Fig. 3B), which constitutes the only difference between the two EM maps of the tail proximal extremity. To increase the number of particles and to achieve better resolution, we combined both EM data sets but it did not lead to any sensible improvement of the map.

## The tail completion protein TCP<sub>p143</sub>

As mentioned in the introduction, TCPs are a highly conserved family of tail proteins, expected to be located in the neck of phages given the position of their genes in the tail module of phage genomes (5–7, see also https://phrogs.lmge.uca.fr/). Phage T5 TCP p143, TCP<sub>p143</sub>, is a 27.8 kDa protein, which gene is sandwiched between that of T5 Head Completion Protein and of TrP<sub>p142</sub> (Fig. 1H). In the cryo-EM map of the T5 tail proximal extremity presented here, we could clearly trace TTP<sub>pb6</sub> trimers and a hexamer of TrP<sub>p142</sub> (Fig. 1E, 3A, and B). Inside the tube, low-resolution density, similar to that present along the whole tube, can reasonably be attributed to TMP<sub>pb2</sub> (see above). Thus, there is no unassigned density in this proximal extremity map of the tail that could account for TCP<sub>p143</sub>.

TCP<sub>p143</sub> is however present in the T5 virion and purified tails, as it was unambiguously detected by mass spectrometry (8, 10). We recently determined the high-resolution cryo-EM structure of the T5 tail tip. In the C3 map, at the base of BHP<sub>pb3</sub>, a faint
density was visible. Intrigued, we calculated an unsymmetrized map, focusing on this
density, and we could indeed solve a bona fide density, otherwise blurred by the imposed
threefold symmetry, located at the base of the BHP<sub>pb3</sub>, in the groove between the closed
tube and the first fibronectin domain (Fig. 4A; Fig. S2A) (10). This density appears only on
one of the BHP<sub>pb3</sub> monomers, which is completely unexpected considering the overall
C3 symmetry of the rest of the tail. Indeed, the only other monomeric protein of the
assembly is the Receptor-Binding Protein pb5, located at the extremity of the straight
fiber (Fig. 1A)(38). The map resolution in this area (6–10 Å) did not allow us to build a
protein model de novo in this density (Fig. S2A); however, the only unlocalized tail protein
remained TCP<sub>p143</sub> (10).

We used Alphafold2 (AF2) (38) to predict TCP<sub>p143</sub> structure and see whether it could
be fitted into the unassigned density. The five AF2 predicted TCP<sub>p143</sub> structures are
rather heterogeneous and exhibit relatively low confidence levels (pLLDT between 30%
and 90%, Fig. 5; Fig. S3A), which could indicate a highly flexible protein. AF2-TCP<sub>p143</sub>
seems however to be composed of two domains (residues 1–80 and 81–255) (Fig. S3B
and C). The global size and shape of some of the AF2 predicted models correspond
to the size and shape of the unattributed density (Fig. 4B) and flexible fitting led to a
convincing fit of the predicted secondary structures (Fig. 4C; PDB as Suppl. file). The fitted
structure is mostly composed of α-helices and loops, with a small β-sheet formed by
three short strands (Fig. 5; Fig. S3A) (10). Thus, we proposed that this unallocated density
indeed belonged to a monomer of TCP<sub>p143</sub>. Even though our results indicate that only
one TCP<sub>p143</sub> copy per phage is present, cryo-EM single particle analysis consisting of the
averaging of a large number of individual particles, we cannot exclude the possibilities of
zero, two, or three TCP<sub>p143</sub> copies being present on T5 tails. If it were the case though,
these occurrences would only account for a minority of particles, as there is no hint of
more than one full occupancy in our unsymmetrized map. We cannot explain yet why
only one TCP<sub>p143</sub> molecule would bind to the T5 tail tip, as the BHP<sub>pb3</sub> trimer follows

<figure><img src="image_7.png" /><figcaption>FIG 4 (A) Isosurface view of an unsymmetrized and unsharpened cryo-EM map of the tail tip (side view). The BHP<sub>pb3</sub> trimer is in yellow and the DTP<sub>pb9</sub> hexamer is in orange. The additional density located on the side of the BHP<sub>pb3</sub> trimer and corresponding to the monomeric protein TCP<sub>p143</sub> is framed in black. (B–D) Fit of an AF2 predicted model in the additional density (B: unmodified AF2 model, C: flexible-fitted AF2 model, D: flexible-fitted AF2 model in the segmented map). Different views (from left to right: front view, side view, top slice, bottom slice) are shown. The model in panel B is colored according to its per-residue pLLDT/confidence measure. Color key interpretation is the following: 100 to 90 = high accuracy expected, 90 to 70 = backbone expected to be correctly modeled, 70 to 50 = low confidence/caution, 50 to 0 = should not be interpreted, a strong predictor of disordered regions.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**FIG 5** Alphafold2 predictions for different phage TCPs, colored according to their per-residue pLLDT (confidence measure), except for the flexible fitted TCP<sub>p143</sub> (purple). The pLLDT color key interpretation is as in Fig. 2. The bottom panel models are rotated horizontally by 90° compared to the ones of the top panel. Uniprot entries: T5 (Q6QGE0), λ (P03731), SPP1 (O48447), HK97 (E9RJ97), TP901-1 (Q77K21), P2 (P36934), and Mu (Q01261).</figcaption></figure>

a C3 symmetry and, at the resolution of our map, no difference in the three potential binding sites could be identified (Fig. S2B through D)(10).

In the EM map of the T5 tail tip after interaction with its receptor FhuA, TCP<sub>p143</sub> is absent (Fig. S2E). This is not surprising given the position of TCP<sub>p143</sub> at the interface between the tip of the tube and the straight fiber, as the large conformational changes induced by receptor binding, inducing the bending of the straight fiber on the side of the tube, completely re-arrange/destroy TCP<sub>p143</sub>-binding site (Fig. S2E). Given the asymmetry induced by TCP<sub>p143</sub> in the tail tip, its presence could determine the direction in which the straight fiber bends. In any case, it would definitely detach upon infection and could play an active role, remaining to be determined, in the infection process.

To see whether the putative TCPs of different phages share more than a common position in the structural gene module in phage genomes, we predicted the structures of some TCPs (Fig. 5). A first striking difference with TCP<sub>p143</sub> is that all structures are predicted with a high level of confidence (pLLDT >90 for most of the protein). A second interesting feature is that, even though varying in length, from 112 residues for TP901-1 TCP to 192 for λ TCP, these predicted structures all share common features, including two side-by-side long α-helices and a small β-sheet, and superimpose very well (Fig. S3D). AF2-TCP<sub>p143</sub> seems to stand out from the other predicted TCPs in terms of structure and length (255 residues), and thus, it could maybe also stand out in terms of location and be located at a different position in the tail.

As far as we know, no structure of a phage TCP has been solved to date, and their function and localization within the tail remain unknown. Literature about TCPs is scarce, contradictory, and does not help to set the case about their localization or function. Indeed, TCP deletion sometimes leads to phages failing to assemble correctly (tail and
capsid are produced but do not assemble) as in myophage P2 (26), siphophages TP901-1 (39), and SPP1 (40). In other phages, like siphophage λ (gpZ) (33) or myophage Mu (27), TCP deletion leads to normal-looking virions that however exhibit much lower infectivity. Such a dramatic functional difference is difficult to explain, especially considering that their predicted structures appear very similar. The different phenotype of TCP deleted mutants however does not stem from a different neck organization between siphophages and myophages, as in TCP less mutants, myophages Mu and P2 behave differently, as well as siphophages SPP1, TP-901-1, and λ. In the case of phages P2, TP901-1, and SPP1, the fact that TCP deleted mutants do not form complete virions suggests that the presence of this protein is a prerequisite to capsid attachment to the tail, and the most straightforward conclusion would be that it is located in the neck region (40). However, the structure of the SPP1 neck has been determined and, as for T5, unambiguously shows that its TCP-gp16.1 is not present (17). This is also the case for λ, in which TCP-gpZ is detected in the tail particle but not localized in the tail structure (18, 19), and for all myophage neck structures solved to date, where there is no trace of a protein that would be a TCP (e.g., 4, 41, 42). In λ, in which full virions are produced in a TCP-deleted mutant, it was suggested that TCP-gpZ allows for the correct DNA insertion into the tail after capsid and tail attachment (43), explaining the lack of infectivity of gpZ-deleted virions. TCP<sub>p143</sub> is a small, monomeric protein, part of a highly symmetrical megadalton-sized complex, with a very unexpected location. Thus, special attention and sophisticated image processing were required to reveal it in the T5 tail. We would like to kindly encourage our colleagues to have a fresh look at their phage tail cryo-EM data, as this protein should also be present in a majority of phage tails. Increasing data would allow us to shed light on this still mysterious protein.

## Conclusion

We present the structure of the proximal extremity of the phage T5 tail. In the densities available, we could trace, as expected, a hexamer of TrP<sub>p142</sub> and a first trimer of TTP<sub>pb6</sub>. In the lumen of the tail tube before interaction with the receptor, densities of much lower resolution are present, which we attribute to TMP<sub>pb2</sub>. As for the many structures of proximal tail extremity or necks of either sipho- or myophages, no remaining densities, even faint, could correspond to the TCP. Instead, we could localize a medium-resolution unattributed density in the unsymmetrized map of the distal extremity of the T5 tail, at the base of a BHP<sub>pb3</sub> monomer, in which a predicted structure of TCP<sub>p143</sub> could be fitted (10). TCPs have been proposed to form a superfamily of neck proteins, based on iterative profile-profile comparison and genome organization analysis (5, 6, see also https://phrogs.lmge.uca.fr/). However, predicted structures of other sipho- and myophages TCPs share the same fold, whereas TCP<sub>p143</sub> seems to stand out in several aspects (predicted structure and proposed location). Given the dispersity of phenotypes of TCP deleted phages, it is thus possible that TCPs adopt different roles and locations within different phages and would, in fact, not correspond to a homogeneous family of proteins.

## MATERIALS AND METHODS

### T5 tail purification

Tails were purified as in references (8, 10). Briefly, an *E. coli* strain F culture was infected with the mutant phage T5D20am30d, which bears an Amber mutation in the major capsid protein gene. After complete cell lysis and DNA digestion by the addition of DNase, cell debris and unlysed cells were removed by low-speed centrifugation, and T5 tails were precipitated by incubation with 0.5 M NaCl and 10% (wt/wt) PEG 6000 overnight at 4°C. After low-speed centrifugation, the pellet was resolubilized in buffer and purified on a glycerol step gradient. The gradient fractions containing the tails were diluted 10 times, and loaded onto an ion exchange column, and the tails were eluted by
a 0–0.5 M NaCl linear gradient. Purified tails were incubated for 30 min with FhuA-loaded nanodiscs in a tail:FhuA-nanodisc ratio of 1:10 (vol/vol) at room temperature (10).

## Cryo-EM sample preparation

Typically, 3.5 µL of T5 tails sample (with or without FhuA-nanodisc) was deposited on a freshly glow discharged (25 mA, 30 s) Cu/Rh 300 mesh Quantifoil R 2/1 EM grids and plunge-frozen in nitrogen-cooled liquid ethane using a ThermoFisher Mark IV Vitrobot device (100% humidity, 20°C, 5 s blotting time, blot force 0).

## EM data acquisition

Respectively, 3,208 and 5,752 micrographs were collected for the tails alone and the FhuA-nanodisc incubated tails, over three different data collections. 40-frame movies were acquired on a ThermoFisher Scientific Titan Krios G3 TEM (European Synchrotron Radiation Facility, Grenoble, France) (44) operated at 300 kV and equipped with a Gatan Quantum energy filter coupled to a Gatan K2 summit direct electron detector. Automated data collection was performed using Thermo Fisher Scientific EPU software, with a typical defocus range of -1.0 to -3.0 µm and a total dose of 40 e/Å² per movie. A nominal magnification of 105.000× was used, resulting in a calibrated pixel size at the specimen level of 1.351 Å. The images used were the same as those used in reference (10) (Table S2).

## EM image processing

### Preprocessing and particle picking

Frame alignment was performed using Motioncor2 (45) keeping, respectively, frames 3 to 30 and 1 to 40 for Tail and Tail, and applying dose weighting. Contrast transfer function parameters were then determined using Gctf (46); manual particle picking was performed with EMAN2/e2helixboxer (47). The picking coordinates were consistently centered on the last visible ring at the proximal extremity of the tail tube, corresponding to the tail terminator p142 hexamer. All subsequent image processing was performed using Relion (versions 3.0 and 3.1) (48) (Table S2).

### Proximal extremity of the tail (Tail and Tail-FhuA)

Tail and Tail-FhuA data were processed identically. After particle extraction (box size of 140 × 140 pixels²), a run of 2D classification was performed to get rid of bad particles (Fig. S1B). A run of 3D classification was then performed, using a cylinder (diameter 90 Å, height 188 Å) as an initial model, at the end of which a small additional fraction of the particles was discarded, ending up with a homogeneous data set of, respectively, 9,953 and 10,701 particles. 3D refinement with masking and C3 symmetry imposed followed, using the best 3D class as an initial model (low-pass filtered at 15 Å) yielded a C3 map of the proximal extremity of the tail, comprising the tail terminator p142 hexamer and the first TTP<sub>pb6</sub> ring. After masking and sharpening, the overall estimated resolution of the maps, respectively, reached 3.88 Å and 4.05 Å (FSC<sub>0.143</sub>) for Tail and Tail-FhuA (Fig. S1D). For both maps, a local resolution map was calculated using Relion built-in local resolution tool (Fig. S1C).

### Tail completion protein TCP<sub>p143</sub>

The full protocol used to obtain the cryo-EM maps for the T5 tip complex is detailed in Linares et al. (10). Here, we detail the steps that are specific to the map shown in this study. After particle extraction under T5 collar and 2D classification, a homogeneous data set of 9,290 particles was obtained. No 3D classification was performed. Using a 15 Å resolution map determined from a previous cryo-EM data collection (unpublished) as an initial model, a C3 reconstruction of the tip was calculated, from the second TTP<sub>pb6</sub> ring to the beginning of the central fiber. Refined particles from the previous reconstruction
were reextracted (box size of 200 pixels by 200 pixels) and recentered on the lower part of BHP<sub>pb3</sub>, on the side of which TCP<sub>p143</sub> is located. After reclassification/selection, symmetry relaxation, and a new run of 3D refinement using suitable masking and no symmetry imposed, a new map of the central part of the tip complex was obtained, where densities for the monomeric TCP<sub>p143</sub> were visible.

## Protein model building

Atomic protein models were built into the two cryo-EM maps using the Coot software (49) by tracing the protein sequence into the densities and were then iteratively refined alternating Coot manual refinement and PHENIX (50) real space refine tool until convergence. For Trp<sub>p142</sub>, the model of the homologous Trp-gpU from siphophage λ (PDB: F3Z2) (1) was rigid-body fitted in the cryo-EM map of the proximal extremity of the tail and was used as a starting point for model building, while for TTP<sub>pb6</sub> models, existing cryo-EM models (PDB: 7QG9) (10) were used as a starting point and were refined into the EM maps. Molprobity (51) was used for model quality assessment (Table S2).

All the predicted structures in this study were generated using AlphaFold2 (52) on ColabFold (https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb) (53). In addition, for TCP<sub>p143</sub>, we fitted the most promising AF2 predicted model into the unsymmetrized tip cryo-EM map, then used a combination of Flex-EM (54)/Namdinator (55) (flexible fitting) and PHENIX (50) (real space refine), iteratively, to obtain a better model for this protein, with a convincing fit of most of its secondary structures.

## ACKNOWLEDGMENTS

We thank Guy Schoehn for the general scientific and instrumentation support; Grégory Effantin for cryo-EM data collection; Emmanuelle Neumann, Daphna Fenel and Séraphine Degroux for technical help; Leandro Estrozi, Ambroise Desfosses, and Benoît Arragain for help and discussion on image analysis; and Aymeric Peuch for help with the use of the EM computing cluster. We acknowledge the European Synchrotron Radiation Facility for the provision of beam time on CM01. This work used the platforms of the Grenoble Instruct-ERIC center (ISBG; UMS 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-05-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). The IBS acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG, CEA).

This research was funded by the Agence Nationale de la Recherche, grant number Project ANR-20-CE11-0023. The electron microscope facility is supported by the Auvergne-Rhône-Alpes Region, the Fondation pour la Recherche Médicale (FRM), the Fonds FEDER, and the GIS-Infrastructures en Biologie Santé et Agronomie (IBiSA).

C.B. conceived the project. R.L. and C.B. prepared the T5 tails and FhuA-nanodiscs used for high-resolution cryo-EM. R.L. optimized cryo-grid preparation, processed the cryo-EM data, built the atomic models, and analyzed and interpreted the models with the help of C.B. R.L. and C.B. wrote the paper.

## AUTHOR AFFILIATION

¹Université Grenoble Alpes, CNRS, CEA, IBS, Grenoble, France

## PRESENT ADDRESS

Romain Linares, European Molecular Biology Laboratory, Grenoble, France
## AUTHOR ORCIDs

Romain Linares ID http://orcid.org/0000-0001-9226-3382

Cécile Breyton ID http://orcid.org/0000-0002-4382-0434

## FUNDING

<table><thead><tr><td>Funder</td><td>Grant(s)</td><td>Author(s)</td></tr></thead><tbody><tr><td>Agence Nationale de la Recherche (ANR)</td><td>Projet ANR-20-CE11-0023</td><td>Cécile Breyton</td></tr></tbody></table>

## AUTHOR CONTRIBUTIONS

Romain Linares, Formal analysis, Methodology, Validation, Writing – original draft, Writing – review and editing | Cécile Breyton, Conceptualization, Funding acquisition, Methodology, Resources, Supervision, Validation, Writing – original draft, Writing – review and editing

## DATA AVAILABILITY

The cryo-EM density maps of the T5 tail proximal extremity presented in this study and the associated atomic coordinates have been, respectively, deposited in the Electron Microscopy Data Bank (EMDB) and Protein Data Bank (PDB) under the following accession codes: EMD-15967/PDB 8BCP (native state) and EMD-15968/PDB 8BCU (after interaction with FhuA). The unsymmetrized cryo-EM density map of the T5 tail tip has been deposited in the EMDB under accession code EMD-51232. TCP<sub>p143</sub> AF2 flexible-fitted model is provided as supplemental material.

## ADDITIONAL FILES

The following material is available online.

## Supplemental Material

Supplemental text (JVI01376-24-s0001.txt). PDB file of TCP<sub>p143</sub> AlphaFold2 predicted-model fitted into the free density of T5 tail tip.

Supplemental material (JVI01376-24-s0002.pdf). Figures S1 to S3; Tables S1 and S2.

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