Peer review of: The architecture and stabilisation of flagellotropic tailed bacteriophages (2020)

Peer Review File, Reviewers' comments first round: 
 
Reviewer #1 (Remarks to the Author): 
 
Comments on “The architecture and stabilisation of flagellotropic tailed bacteriophages” 
 
The authors present combined structural methods including cryoEM, crystallography, and bioSAXS 
to provide novel insights into the structure and potential assembly of the previously poorly 
characterized but fascinating flagellotropic phage family, specifically the non-contractile tails 
therein. The resultant data delineate clearly the structural features underlying assembly and 
suggest reasons that these structures are able to withstand the substantial shear forces 
experienced when bound to the rotary extracellular flagellar appendages of their bacterial host. 
The model of DNA passage is compelling and a foundation for further studies probing 
understanding of rapid passage of these large genomic substrates from phage to the bacteria they 
infect. Phage reagents are a hot bed of potential therapeutic applications currently and collectively 
I believe this work will be of significant and broad interest to those studying protein assemblies 
and infection. 
 
Line 76 – “Recent genome sequence analysis had indicated that, at a protein structural level, YSD1 
and χ-phage virions are indistinguishable22” –had to check the ref to see that they mean the 
protein seq would be 99-100% identical (however structures can be highly similar even with 
disparate sequence). This should be rephrased for clarity. 
 
Line 78 and throughout the manuscript please define resolution values as such “3.8 Ang structure” 
should be “3.8 Ang resolution structure” 
 
Figure S1a – please provide a scale in the negative stain image as well as a brief mention in the 
methods (stain type etc). 
 
Line 129, add resolution of the crystal structure here. Why is the crystal structure captured as a 
monomer? Please specify number in the au in the methods as well as any potential crystal packing 
interfaces of relevance. Are any mimicking that of the cryo EM assembly? Or alternatively any 
potential influence of the observed structure from crystal packing? 
 
Line 154, cyan should be magenta? 
 
Line 167, here and throughout the manuscript – please provide the number of atoms used in the 
rmsd calculation 
 
Line 220; the values to the hundredths place given the potential coordinate error at this resolution 
are likely unwarranted. 
 
Line 329 – “The β-hairpin, the N-terminal arm and loops on the head-proximal side of the 
hexameric ring protrude from the SAXS envelope (Fig. 7a) suggesting that these structural 
features are either flexible or folded back onto the core structure” – I think that these are certainly 
options, but even if these small extensions were rigid, I’m not sure that density would be visible 
within the resolution scope of bioSAXS. In general, although the model of assembly is compelling, 
I think the statements in this section (lines 328-355) are reaching beyond the scope of the data. 
Some mutagenesis or additional experimentation could be nice in this regard to solidify this 
proposed assembly. The comment above also applies again in the Discussion (lines 395-397), and 
Figure 7. 
 
Why was RELION2 used for the processing? It might be appropriate to reprocess in RELION3.1, 
particularly to see if any additional insight can be pulled from the region of the helical tail that is 
connecting to D3. 
 
A SAXS stats table is not present in the supplement – some modified form of what is in the 
guidelines here: https://journals.iucr.org/d/issues/2017/09/00/jc5010/ would be appropriate. 
 

Throughout: capsomer should be capsomere? 
 
 
 
Reviewer #2 (Remarks to the Author): 
 
The MS “The architecture and stabilisation of flagellotropic tailed bacteriophages” describes high 
resolution structure of bacteriophage YSD1 that infects Salmonella Typhi. YSD1 is similar to phage 
Chi, which is known to attach to the bacterium flagellum. The MS contains a substantial number of 
bold statements, but most of them, unfortunately, remain unsupported by the findings. For this 
reason, the MS needs to be substantially modified to become an actual article. One positive aspect 
of the MS is that the figures are absolutely beautiful. 
 
Please find my line-by-line comments below. 
 
The abstract 
 
- However, it is unclear how tailed bacteriophages withstand the substantial rotary shear forces 
generated as they move down the spinning flagellum. 
I am not sure why the “rotary shear forces” are singled out here. Are the “shear forces” acting on 
the particle attached to cell surface lipopolysaccharides with its tail fibers weaker? 
 
- In addition, the structure of the tail reveals concerted rearrangements 
A supporting evidence for concerted rearrangement is not presented in this paper. 
 
- provide an elegant means for promoting genome translocation 
To claim that something promotes something, you need to show evidence that in the absence of 
this feature the process in inhibited. 
 
The Intro 
 
- In contractile tails of myoviruses, an outer sheath adds to the stability of the central tail-tube, 
however it is unclear how tail-tube integrity is maintained in flexible tails found in the majority of 
phages. 
What is the basis for this remarkable statement? Myophage tail tubes are pretty stable in solution 
(although the baseplate is required for assembly). In the contracted state, about half of the tube 
protrudes below the plane of the baseplate. The sheath does NOT interact with the tube in this 
state at all, so the sheath is not required to maintain the stability of the tube for DNA 
translocation. You really need to read some literature… 
 
- The best available model of such a tail is derived from a pseudo-atomic model for phage T5, 
which has an atypical 3-fold symmetry. 
The structure is nearly perfectly 6-fold symmetric. There is no need to bash it for being 3-fold. 
 
- In the absence of a contractile sheath, how the ~10 µm (~60 kbp) genome efficiently transits 
through this long narrow tunnel is also not fully understood. 
Again, this is a remarkable statement in its erroneousness. DNA translocation and sheath 
contraction are not linked. 
  
- and four classes of chainmail 
I think there is only one type of chainmail, which is actually cross-linked. All other cases of capsid 
stabilizations are not chainmail. I believe there are only three fundamentally different ways to 
stabilize the capsid: 1) covalent cross-linking of capsomers (chainmail), 2) additional domains to 
the HK97-fold and 3) additional decoration proteins. 1), 2) and 3) can exist in different 
combinations (e.g. 2) + 3) or 1) +3), etc.). Prolate or not has nothing to do with “chainmail 
stabilization” (which is an incorrect concept as explained above) as far as existing data show. 
 
- (ii) the P22-like capsids that are stabilised by an inserted domain in the minimal HK97 fold19; 
The term “insertion domain” was introduced a few years before Kristin Parent’s publication 

(PMID: 15878991). It is a pity that Kristin failed to acknowledge that the insertion domain in the 
P22 capsid protein is located at the same position as the insertion domain in T4 gp23/gp24 
(compare Fig. 2 from Fokine at al PNAS 2005 and Fig. 5A from Parent et al Structure 2010). And I 
find it even more disappointing that the discovery of the insertion domain and its possible role in 
capsid stabilization has not been attributed to the original authors in this manuscript. 
 
The Results 
The internal surface of the tail-tube facilitates DNA ejection 
- Given the conservation of similar acidic residues dyads in the T5 crystal structure and sequences 
of tail-tube proteins, we suggest that this DNA translocation mechanism is general to all 
Siphoviridae. 
Are those residues conserved? Can you please support this statement by showing sequence 
conservation mapped onto the structure? 
 
 
Assembly of a flexible yet robust tail-tube 
- These changes would mask the specific regions that form the inter-ring interfaces in the 
assembled tail. In a model of nucleation of the tail-tube polymerisation summarised in Figure 7, 
the action of the initiator would thus require splaying of the β-hairpin and N-terminal arm to allow 
clipping of tail-tube protein subunits into the hexameric ring. 
So, we cannot see a density for the hairpin or the N-terminal arm in the SAXS envelope and the 
conclusion is that the two are folded back and interact with each other? This conclusion needs to 
be supported by additional experiments such as cross-linking or mutagenesis that would show that 
the two elements interact in the recombinant protein. As this finding is central to the later stated 
hypothesis, the hypothesis has a very shaky foundation. 
Discussion 
 
 
- These features raise three major questions addressed in this study: what induces the highly 
soluble precursors to polymerise into the tube, how is stability achieved in a flexible helical 
structure, and how does the structure promote DNA translocation? 
This study reported the structure and atomic models of the capsid and tail tube of phage YSD1. 
The first question is about particle morphogenesis and it cannot be answered with the structure 
data alone. It requires hypotheses, mutagenesis and analysis of assembly intermediates. The 
second question is about stability. To claim stability, the actual stability needs to be measured. 
Mutants have to be created and their stability needs to be measured. The third question is about 
DNA translocation. Finding a negatively charged channel tells un nothing about the mechanism. 
The portal protein of phage phi29 was the first phage-derived negatively charged channel. Please 
show how the YSD1 is different. What do those regularly spaced residues in the YSD1 channel do? 
 
- Here we show that to form the hexameric building blocks of the tail, the β-hairpin and N-terminal 
tail need to unmask complementary hydrophobic regions in the tail tube protein. 
Please see my earlier remarks about the actual evidence of the claimed property. 
 
- More than this, the precise arrangement of negative charges revealed here suggests a 
mechanistic explanation for the theoretical need for DNA to ratchet from the tube once the initial 
impetus to the DNA from the pressurized environment of the capsid begins to wane. 
I am not sure that I follow the logics. The tube is negatively charged. It contains regularly spaced 
negatively charged amino acids as it should (it is a repetitive polymeric structure). All that repels 
the DNA and does not let it stick to the side of the tube. What ratchets are we discussing here?