Peer review of: The structures of two archaeal type IV pili illuminate evolutionary relationships (2020)

Reviewer #1 (Remarks to the Author): 

Wang et al present two new archaeal type 3 pili (T4P) structures determined by cryo-EM, which 
add to the single presently known archaeal T4P structure and enables them to compare all known 
structures of bacterial and archaeal T4P structure with each other and archaeal flagellar filaments. 
They identify glycosylation as a key factor for the high thermal stability of T4P and describe their 
interaction with helical viruses. Without leaning on prior structural information, they remarkably 
succeeded by sequencing the genomes of these archaea and by combining mass spec and 
bioinformatics to build de-novo models into the cryo-EM maps. Combining these multi-disciplinary 
analyses, the authors propose an intriguing hypothesis about a common evolutionary origin 
between T4P and archaeal filaments. 

This is an excellent paper, well-written and packed with clearly arranged dense information. I have 
thoroughly enjoyed reading it. The figures are excellent. The presentation is logical and easy to 
follow. It is one of the rare cases I recommend publishing without any significant further 
modifications. 

Minor comments: 

Line 439: “b”, use bold face 

Figs. 4d and 5a: I really like these. Well done! 

Line 308: “The helical symmetry was determined in SPIDER using IHRSR after searching through 
all possible symmetries by trial and error”. This statement should be expanded to explain the 
search strategy more comprehensively. There are many local maxima in helical symmetry 
searches and it would be computationally expensive to explore them all. At the minimum, it should 
be stated that a local search around the helical symmetry shared by the known archaeal filament 
structures yielded a solution that refined to high resolution. 

Lines 300ff: this paragraph apparently contains some unprintable characters (e.g. 300?keV) 

Lines 256-258: I don’t follow the argument why the NTD would have evolved first. Please clarify. 

Line 248: “suggesting that it is glycosylation” -> tone down a notch to “suggesting that it may be 
glycosylation” 

Signed: M.Wolf 


Reviewer #2 (Remarks to the Author): 

This well written paper by Wang et al. presents structures of two archaeal T4Ps from P. 
arsenaticum, a neutrophilic hyperthermophile and S. solfataricus, an acidophilic hyperthermophile, 
after co-purification with their respective viral partners and characterization by cryo-electron 
microscopy (cryo-EM) to identify the relevant gene product among multiple possibilities as they 
have done previously. The authors compare these structures to other filaments from the same 
superfamily to understand some of the distinguishing characteristics. Comparisons of the amount 
of glycosylation were used to explain differences in stability of these filaments in harsh conditions, 
particularly low pH. The This work adds two more structures of archaeal T4Ps for structural 
comparisons to explain evolutionary lineages, including more confident assignment of Iho670 
adhesion filaments as T4P. Despite the use of phage to identify the relevant filaments, the 
potential role that glycosylation plays in affecting the viral-T4P interaction was not addressed. Do 
the phages bind to the archaeal pilins directly or to the sugars? The three themes of viral-host 
interaction, glycosylation of the two T4Ps and structural comparisons of the archaeal T4Ps with 
bacterial T4Ps and archaeal flagella could be better connected. 

Comments: 
Line 59, ectodomain is an odd term to use 

Line 103, too many significant figures. The opposite 7-start nomenclature has the potential to be 
confusing to readers not familiar with this filament family, especially since both are right handed 
one start helices. 

Line 152, are pilin glycosylation enzymes encoded with the pilin genes? 
In the third paragraph of results, the authors mention that the C-terminal domains are structurally 
conserved, except “one β-strand in P. arsenaticum maps to two shorter β-strands in S. 
solfataricus”. What is the functional significance of this difference, is it conserved in each group of 
archaea? 

Line 188, electrostatic interactions 

Line 230, Tad pili extend and retract with only a single ATPase (shown by the Brun lab for 
Caulobacter), this is not unprecedented 

Line 246, disulfide bonds are common in bacterial type IV pilins, both at the C-terminus and 
internal in some species – this should be mentioned, as written it sounds more unique than it is 

Line 255, I don’t understand this statement based on the preceding evidence 

Figure 2c descriptions: specify the sequence alignment program used and what the different 
shades of blue correspond to in the alignment map 

Figure 2e: use arrows to represent β-sheets instead of circles 

Figure 2a/b/f: indicate the N and C-termini 

Many comparisons were made between S. solfataricus/S. islandicus and P. arsenaticum pilins 
including % of charged residues, O-/N-linked glycosylation sites, sequence identities, etc. To help 
readers appreciate these differences, a table or schematic showing side-by-side comparisons of 
these parameters would help 

Figure 3 legend descriptions of panels a and b are switched 

Figure 3a: what do blue, orange, green represent? 

Supplementary figure legends 3 and 4 are switched 

In the seventh paragraph of results, figure identification should be (Fig. 4a,c) instead of (Fig. 3a,c) 

In the third paragraph of discussion, the supplementary figure was incorrectly labelled. Should be 
(Supp. Fig. 4) not (Supp. Fig. 3) 

Is it necessary to include subheadings for each of the results? 



Reviewer #3 (Remarks to the Author): 
In their manuscript Wang et al. report two new cryo-EM structures of archaeal Type IV pili, that of 
Sulfolobus islandicus and Pyrobaculum arsenaticum, and demonstrate these structures represent 
the receptors for two filamentous phages recently identified by the authors. The new T4P 
structures allow a robust analysis of the phylogenetic relationship and structure-function aspects of 
bacterial and archaeal T4P as well as the T4P-related archaeal flagellum, also known as 
“archaellum”. In addition, the authors discuss the role of T4P glycosylation in function of the 
chemical and high temperature resistance of the fibers. 

The paper is well written and brings new insights in the evolutionary relationships of T4P-like 
filaments that should be of interest to a diverge audience. 

Overall, the paper is technically sound and the data are supportive of the conclusions presented, 
with the exception of the proposed claims regarding the high stability of the fibers, which would 
require some additional experiment to be conclusive. This reflects the single major comments I 
have, which is the lack of systematic experimental interrogation of the structural aspects that 
provide the fibers’ strength. There is no direct demonstration that the glycosylation or the C-
terminal extension in T4P of S. solfataricus and P. arsenaticum provides the fibers’ resistance to 
acidic or high temperature conditions, respectively. The only data shown is qualitative in nature 
(Suppl. Fig. 4) and shows single negative stain EM images only. A more quantitative and 
comprehensive analysis of the chemical and heat stability of the fibers would be advisable. For 
example by monitoring the release – or lack thereof – of subunits by SDS-PAGE. 

Specific comments and suggestions: 
1. In Ln 90-100 and Figure 1 the authors convincingly demonstrate that T4P pili represent the 
receptor for filamentous phages SSRV1 and PFV2. Presumably these interactions are specific for 
the cognate T4P – phage pair? It would be good to evaluate this and mention it in the paper. 
In the discussion the authors propose that the targeting of T4P by the phages may be related to 
the strong conservation of the pili or a possibility for the phages to move along the length of the 
fiber to gain access to the cell. Another aspect worth considering is that T4P may represent a 
location corresponding to a breach in the S-layer, which may facilitate access to the cytoplasmic 
membrane. 
2. In lines 118-128 the authors derive and discuss the cleavage sites of the signal peptides in 
prepilins. It would be worth highlighting this finding in an additional figure panel. For example as 
part of Suppl. Figure 2. 
3. Lines 131-136 and in Fig 2e, the authors compare structural similarity in the T4P N- and C-
terminal domains. It would be helpful to provide RMSD values for the alignment of the respective 
domains to give some quantitative measure of the similarity. 
4. In Ln 139-142 and Fig. 2f the authors describe the presence of a disulfide-bonded C-terminal 
extension in P. arsenaticum. It would be helpful to also illustrate the location of the disulfide linked 
C-terminal extension in the context of the full fiber. Is there a possibility to form intermolecular 
crosslinks amongst subunits, or does the structure only allow for intramolecular crosslinking? 
5. In ln 144-147 S. solfataricus and S. islandicus pili are said to have 37% Ser/Thr in the globular 
domains. This does not seem to correspond with the data shown in Fig. 3a, which appears to show 
more 30-31% in Ser/Gly. Please correct or clarify the difference. 
6. In ln 149-160 the authors describe the indications of glycosylation in the two T4P structures 
solved. It would be good to mention whether glycosylation is also seen in the MS fingerprint 
peptides, and if not, why. 
7. Unless for my oversight, I did not find Suppl. Figure 3 cited in the main text, and in line 165, 
“Suppl. Fig. 3” should be “Suppl. Fig. 4). Also not that in Figure 3, the caption of panels a and b 
does not correspond with the order of the panels in the Figure. 
8. In Jarrel and Albers 2012 (ref https://doi.org/10.1016/j.tim.2012.04.007) the name 
“archaellum” was proposed for the “archaeal flagellar filaments”. This reviewer is of the opinion 
that that proposed nomenclature does well in highlighting the fundamental difference in bacterial 
and archaeal flagella, and would advise the authors to adopt the nomenclature in their manuscript.