There is limited information about the potential individual-level and contextual drivers of heavy drinking in South Africa. This study aimed to identify risk factors for heavy drinking in Tshwane, South Africa.A household survey using a multi-stage stratified cluster random sampling design. Complete consumption and income data were available on 713 adults. Heavy drinking was defined as consuming ≥120 ml (96 g) of absolute alcohol (AA) for men and ≥ 90 ml (72 g) AA for women at any location at least monthly.53% of the sample were heavy drinkers. Bivariate analyses revealed that heavy drinking differed by marital status, primary drinking location, and container size. Using simple logistic regression, only cider consumption was found to lower the odds of heavy drinking. Persons who primarily drank in someone else’s home, nightclubs, and sports clubs had increased odds of heavy drinking. Using multiple logistic regression and adjusting for marital status and primary container size, single persons were found to have substantially higher odds of heavy drinking. Persons who drank their primary beverage from above average-sized containers at their primary location had 7.9 times the odds of heavy drinking as compared to persons who drank from average-sized containers. Some significant associations between heavy drinking and age, race, and income were found for certain beverages.Rates of heavy drinking were higher than expected giving impetus to various alcohol policy reforms under consideration in South Africa. Better labeling of the alcohol content of different containers is needed together with limiting production, marketing and serving of alcohol in large containers. In 2011, South African adults (aged 15 years and older) consumed 9.5 l of absolute alcohol each year -- higher than the average for Africa 6.0 l) and the world (6.2 l) –6. In ad.0 l and Drinking patterns shape the association between alcohol consumption and related harms, because they determine the dose of toxic effects (which cause chronic disease) and the level of intoxication  . Many alHeavy drinking is a pattern of consumption involving consuming large volumes of alcohol during one occasion or in a short period of time. There is no universal definition for heavy drinking, but studies generally define it using one of two thresholds: 1) 60 g of absolute alcohol for men and 40 g for women or 2) 100 g of absolute alcohol for men and 60 g for women . While tMonitoring heavy drinking trends over time may help researchers predict treatment and prevention needs at the population level. In addition, drinking patterns modify the association between per capita consumption and related harms such that countries with riskier drinking patterns tend to experience increased levels of harms from increases in consumption . This imSouth Africa is currently considering a liquor amendment bill to reduce per capita consumption, including provisions that would raise the national minimum legal purchase age from 18 to 21 years, establish a minimum 500-m buffer between alcohol outlets and other outlets or sensitive locations , and hold alcohol manufacturers and suppliers of alcohol to unlicensed alcohol outlets liable for damages resulting from consumption of their products. The proposed bill also brings some informally and illicitly produced alcohol into the regulated sector by lowering the threshold used to define alcoholic beverages from 1.0% alcohol to 0.5% alcohol .However, there is an incomplete picture of heavy drinkers in South Africa. To date, the literature begins to assemble demographic profiles of heavy drinkers: they tend to be young, male, Black African or Coloured, and reside in urban areas , 22. HowData for this study are from the South African arm of the multi-country International Alcohol Control (IAC) study . This crThe study used a multi-stage stratified cluster random sampling design. There were four stages to the cluster random sampling involving sampling of wards (Stage 1), enumeration areas (EAs) within selected wards (Stage 2), households within selected EAs (Stage 3), and study participants within selected households (Stage 4). This is described in detail elsewhere . Data weWe adapted the standard (English) IAC questionnaire, then translated and back-translated it into seTswana and Afrikaans. It included various items, with those relevant to this paper being demographic factors  and alcohol consumption.Participants’ ages were categorized as: 18–19, 20–24, 25–34, 35–44, 45–54, and 55–65 (reference group). Annual personal income was categorized into low , medium , and high .Heavy drinking was defined as consuming 96 g of absolute alcohol (AA) or more  for men or 72 g or more  for women at any location at least monthly. This definition, used by the IAC study , is highEach typical drinking occasion at each location was classified as low risk or heavy drinking based on the usual quantity of alcohol consumed at least monthly.Occasions that did not include heavy drinking (96 g AA for men and 72 g AA for women) were defined as low risk.Primary drinking location was defined as the location in which the participant reported drinking most frequently. If the participant drank at two locations with the same maximum frequency, then the location where the participant consumed a greater quantity of absolute alcohol was selected. If there were two locations with the same maximum frequency and quantity, then the more exotic location was selected . The primary drinking location variable was categorical with 12 of the 16 original drinking locations included: own home, someone else’s home, nightclubs, sports clubs, other clubs, restaurants, motor, sports events, outdoors, shebeen, pub, hotels, special events, and other. No participants primarily drank in theaters, planes, workplaces, hotels, or at sports events.The primary beverage consumed at the primary drinking location was selected by determining the beverage the participant drank with maximum quantity (of AA) at that location. The primary beverage variable was categorical with 12 of the original 14 beverage types: beer; low alcohol beer; home brew beer; stout; wine; spirits; cocktails; liqueur; shooters; sherry, port, or vermouth; cider; alcopops , and other beverages. No participants primarily drank other beverages or sherry, port, or vermouth.Container size was determined as the usual container size of the primary beverage at the primary drinking location, and was categorized into average, below average, or above average. Average container size was defined as the container size closest to a standard drink .After obtaining informed consent, participants were interviewed in their homes by trained interviewers. Interviews were administered on a tablet. This approach was adopted due to the complexity of the questionnaire. After the interview, participants received a resource card for alcohol-related problems as well as a shopping or a cellular telephone recharge voucher worth R30. The study was approved by the Research Ethics Committee of the South African Medical Research Council.Taylor series linearization approximations  were use> 0.5. Model fit was checked using an adaptation of Hosmer Lemeshow’s Goodness of Fit Test, and all models indicated appropriate fit. P values less than 0.05 were considered statistically significant.The analysis then used multivariate logistic regression to test the hypotheses that heavy drinking differs by demographics and alcohol consumption characteristics. Variables with significant relationships to the outcome variables and key demographic variables  were selected using best subset variable selection methods with no variables forced into the model. The model included age, race/ethnicity, marital status, and container size. While other variables explained variability better than primary beverage in the main model, simple logistic regressions of heavy drinking on primary beverage type and primary drinking location were performed to investigate the impact of beverage and location choices. The multiple logistic regression model was then repeated for the persons who consumed the four main types of alcohol  to determine whether the associations differed by beverage. Multicollinearity was assessed by examining correlations between predictors. No two predictors had a correlation Nine hundred and eighty-seven participants did not report frequency data for all drinking locations, and seven participants did not report enough consumption information for their primary drinking location to determine primary beverage and/or primary container size. In addition, 449 participants did not provide a total annual personal income. These participants were excluded from the analyses. The final sample size included 713 adults.F2.06, 39.22 = 2.18, p = 0.12), income , or urbanicity . Participants with missing consumption data were more likely to be younger  and female , and missingness differed by marital status . Participants with missing personal income data did not differ on gender , urbanicity , heavy drinking status , primary beverage , primary drinking location , or primary beverage container size . Participants with missing personal income data were more likely to be younger  and less likely to be Black African , and missingness differed by marital status .Participants with missing consumption data did not differ from the sample on race/ethnicity , age , race/ethnicity  or total annual personal income . Heavy drinking differed by marital status .The mean age in the sample was 36.3 years, 65.8% were male, 79.1% were Black African, and 77.0% were low-income . Among the commonly reported primary drinking locations, persons who primarily drank at special events (91.8%), in motor vehicles (87.2), other clubs (77.5), nightclubs (79.8%), shebeens (61.5%), someone else’s home (65.8%), and pubs (57.3%) had the highest percentages of heavy drinking. Persons who primarily drank at restaurants (19.5%) had the lowest percentages of heavy drinking.Homes (59.9%), pubs (15.3%), someone else’s home (14.8%), nightclubs (2.2%), outdoors (1.9%), shebeens (1.7%), other clubs (1.3%), and restaurants (1.1%) were the most common primary drinking locations. Heavy drinking differed by primary drinking location . High percentages of persons who primarily drank beer (62.5%), wine (50.7%), cider (45.8%), and spirits (42.7%) were heavy drinkers.Beer (48.4%), cider (17.9%), wine (14.4%), and spirits (12.6%) were the most commonly reported primary beverages consumed at the primary drinking location. Heavy drinking did not differ by primary beverage . Fifty-eight percent of the sample primarily drank from above-average sized containers, 34.0% drank from average-sized containers, and 7.7% drank from below average-sized containers. Seventy-two percent of persons who drank from above average-sized containers were heavy drinkers, while only 26.7% of persons who drank from average-sized, and 19.0% of persons who drank from below average-sized containers were heavy drinkers.The container size of the primary beverage at the primary drinking location was also associated with heavy drinking , but other studies conducted among drinkers in South Africa, have similar levels of risky drinking at weekends among male and female drinkers [A large proportion (53%) of drinkers in Tshwane Metropole, South Africa drank heavily (70% of men and 30% of women), even when using a conservative definition of heavy drinking. These heavy drinkers drank the vast majority (93.9%) of the absolute alcohol sold. Primary beverage container size emerged as having the most consistent association with heavy drinking, and it held across four of the most common beverage types. Drinkers who primarily drank from above average-sized containers had nearly 8 times the odds of heavy drinking compared to persons who primarily drank from average-sized containers after adjusting for demographics like age, sex/gender, race/ethnicity and income level. Surprisingly heavy drinking did not differ by gender , 93% of the absolute alcohol was consumed during heavy drinking occasions in Tshwane, South Africa . South AThe results from this analysis also imply that there is another important contextualizing factor at play in the South African drinking environment: the alcohol industry. Our finding that 93.9% of the absolute alcohol is consumed by heavy drinkers in the Tshwane Metropole suggests that the alcohol industry’s revenues in South Africa depend on heavy drinking. The alcohol industry often argues that alcohol-related problems only affect a subset of drinkers and the majority of drinkers consume alcohol “responsibly” , but theAnother key finding from this study is the strong association between primary container size and heavy drinking in Tshwane Metropole. This suggests that container size may promote risky drinking and that the liquor industry may well be driving heavy drinking and hence be a major contributor to alcohol’s high burden of death and disability in South Africa. Beer containers in South Africa often contain two or more standard drinks . In 2017, South African Breweries introduced a larger version (500 ml) of Carling Black Label beer, and priced it the same as their previous 440 ml version . That saDespite the IAC’s innovative survey design, this analysis has limitations. These data are specific to Tshwane, and may not generalize to other parts of South Africa. The analyses only included data from adult participants, so the results may not generalize to youth drinkers. Future research should expand to other areas in South Africa to determine whether these trends are local, regional, or national, and should include youth to detect age-related trends early in life. Data for this study are cross-sectional, so we are unable to rule out reverse causation, which would suggest, for example, that heavy drinkers choose extra-large containers.The definition of heavy drinking used in this study is extreme. Considering that the threshold of heavy drinking is designed to separate drinkers who cause/experience harm from others, such a definition may result in false negatives. However, this choice in operational definitions also increases the likelihood that the drinkers identified as problematic using this definition really are suffering/causing alcohol-related problems. Relatedly, the IAC questionnaires ask about “typical” drinking occasions, and it is possible that respondents overestimate the amount of alcohol consumed when they average across drinking events. However, this analysis capped reports of alcohol consumption at roughly 2500 g of absolute alcohol, which would limit the effects of any extreme overestimation. Further, the previous studies that demonstrate the IAC has high coverage of alcohol sales  suggestsFinally, a sizable portion of respondents had missing data and were excluded from the analysis. The net effect of this missingness may have been to increase the width of some of our confidence intervals, rendering some differences insignificant. This may explain non-significant findings for differences in heavy drinking prevalence by sex/gender, and in the odds of heavy drinking in the simple linear regression for beverage type and the multiple regressions that stratified by beverage type.Heavy drinking is common among current drinkers in South Africa, and heavy drinkers consume most of the alcohol sold. Primary container size emerged as the most robust correlate of heavy drinking. South Africa is currently contemplating alcohol policy reform, and this study underscores the importance of these draft policies. The draft liquor amendment bill of 2016 proposes several evidence-based policies that could help reduce these heavy drinking occasions. Rigorous monitoring of the heavy drinking environment may also serve to establish baseline data to evaluate the effects of any future policy changes.


Caudovirales) and herpesviruses form capsids by a mechanism that includes active packaging of the dsDNA genome into a precursor procapsid, followed by expansion and stabilization of the capsid. These viruses have evolved diverse strategies to fortify their capsids, such as non-covalent binding of auxiliary ‘decoration’ (Dec) proteins. The Dec protein from the P22-like phage L has a highly unusual binding strategy that distinguishes between nearly identical three-fold and quasi-three-fold sites of the icosahedral capsid. Cryo-electron microscopy and three-dimensional image reconstruction were employed to determine the structure of native phage L particles. NMR was used to determine the structure/dynamics of Dec in solution. The NMR structure and the cryo-EM density envelope were combined to build a model of the capsid-bound Dec trimer. Key regions that modulate the binding interface were verified by site-directed mutagenesis.The major coat proteins of dsDNA tailed phages (order  Caudovirales, the capsids use a T = 7 geometry  within the capsids, resulting in high-energy states that prime the particles for infection, and facilitate delivery of the majority of the viral genomes into the hosts . Phages Podoviridae), in the sub-group of P22-like phages  the potential exploitation of phages in nanomedicine , (2) strOB-fold; , and theAs a first step toward understanding the mechanism by which Dec binds to phage L, we determined the structure of native phage L capsids using cryo-EM . Local rAs an initial guide for accurately fitting the phage L coat protein into the cryo-EM density, we used the most recent 3.3 Å-resolution structure of phage P22 coat protein . The sites with the largest R2EX contributions are similar to those with low S2 order parameters, in particular loops L23, L45 and the region surrounding the loop L3α  into the segmented cryo-EM density, independently fitting three copies of the OB-fold. Using Phenix, each copy was refined by flexible fitting to accommodate the slight differences in the asymmetric trimer density. We then built a complete trimer by using a combination of homology modeling and flexible fitting to refine the C-terminal portion of Dec separately from that of the OB-fold. Sequence homology of the C-terminus most closely matched a fragment from the bacteriophage T4 trimeric proximal long tail fiber protein gp34 , so that any phages produced are the result of complementation by the coat gene expressed from the plasmid. None of the amino acid substitutions affected virion production or infectivity; phages assembled with each coat protein variant were grown to high titer and were phenotypically indistinguishable from the parental phage.Previous work suggested that Dec binding to P22 capsids is driven primarily through electrostatic interactions , and ourCoat proteins occupying different local conformations  contribute different residues to the binding interface , Video 3Among the five coat protein changes, coat amino acid substitutions E81R and E323R attenuated but did not completely abolish Dec-binding ability . We creaThe close contact region between Dec and coat protein can be thought of as including two binding sites. One site includes coat residues E81 and P82 that interact with Dec residues K30, Y31 and Y49  were used for all experiments.Phage L, its bacterial host, and purification procedures to produce high-titer stocks were previously described . Stocks Salmonella enterica serovar Typhimurium strain DB7136 , expressing P22 coat protein from mutant pMS11 plasmids, were infected with P22 phage carrying amber mutations in gene five to stop production of phage-encoded coat protein as described previously (eviously . This P2eviously .R (100 μg/mL) and IPTG inducible (1 mM). Sequences were confirmed at the RSTF Genomics Core at Michigan State University. Plasmid pDec was mutated using Quikchange protocols, and was used to express wild type and variant Dec, each with a C-terminal histidine tag. Phage P22 purification  of phage L virions were vitrified and examined using published procedures . Briefly, v2.7.1  on entirhttp://cryoEM.ucsd.edu/programs.shtm) was used to estimate micrograph defocus and astigmatism, extract individual phage L particles, and to preprocess the images. 150 particle images were used as input to the random-model computation procedure to generate an initial 3D density map at ~25 Å resolution (0.5) ‘gold standard’ criterion was used to estimate the resolution of the final 3D reconstructions  with the accession number 27435.Samples of recombinant Dec enriched in escribed . To obtaion data . All NMRion data  and have15N- and 13C-NOESY experiments collected on 15N/13C-labeled Dec samples, with or without 50% fractional deuteration. The NOESY experiments were collected on an 800 MHz spectrometer equipped with a cryogenic probe, using a mixing time of 100 ms. Hydrogen bond restraints were included based on H-bond donors and acceptors identified in a long-range HNCO experiment  The stated resolution of the cryo-EM map is ambiguous and misleading. The authors report a 4.2 Å overall resolution of the icosahedral reconstruction . But from Figure 1C, one can clearly tell the map resolution for the Dec protein part is probably around 8~15 Å, much worse than 4.2 Å. Also, the half-map FSC is measuring self-agreement between two maps rather than actual resolution. Therefore the authors need to measure the Model:Map FSC for the capsid proteins and Dec separately, and include those numbers in the paper.2) The features in this cryo-EM map do not look like a typical 4.2 Å map. For example, one usually sees side chain densities on a helix and β-strand separation at this resolution. But these were not seen in the map provided by the authors. Therefore, it is either the authors over-estimated the cryo-EM resolution, or the map has not been properly sharpened. With the map quality as seen, it is hard to believe the detailed amino acid site analysis presented in Figure 5. This also suggests that some of the mutagenesis results can simply be explained by an inaccurate atomic model.3) Both the capsid protein and Dec models sent from the authors have poor protein geometries, meaning they were poorly refined. Both models even have cis-peptides in the structure, which is unacceptable. The authors need to put a table in the main text listing protein geometries  of the deposited cryo-EM models and maps, and those statistics need to reach the common standard of the field.4) One of the most interesting points in the paper involves the Dec selectivity on the Phage L capsid. But why were the mutagenesis experiments done using Phage P22, which doesn't have Dec?5) The structural basis for the selective binding of Dec to capsids was poorly discussed. The MD-based simulations are not helpful in elucidating the mechanism. First, the authors need to understand that the hexons in the capsid do not necessarily have C6 symmetry. This is true from the model sent by the authors, and this means the "quasi-three-fold" and "true three-fold" may have different surface interfaces to Dec, which is the simplest explanation for Dec selectivity. With a 4.2 Å map, the author should be able to compare the surface using real cryo-EM map/models, rather than some MD simulations.Reviewer #2:The manuscript "How phage L capsid decoration protein distinguishes between nearly identical binding sites on an icosahedral virion" by Newcomer et al. looks at the binding of Dec on the capsid exterior surface in its host phage L, which varies according to position on the capsid surface. In addition, Dec binds to phage P22 due to 99.6% identity in coat protein sequence with that of phage L, although P22 doesn't encode its own Dec homolog. Capsid structures determined by high-resolution cryo-electron microscopy are combined with an NMR solution structure of monomeric Dec, MD simulations, and mutations across the P22 core and Dec interface to reveal the selectivity of Dec binding. Although this is a rather specific example from one of many similar phages, it nonetheless reflects a more general question of the fidelity of protein-protein binding that can also be found in other cellular contexts and for regulating function. Thus, it's a useful illustration of a general process that is readily studied in the tractable context of high-symmetry phage particles that are easily expressed and purified. However, while identifying several residues across multiple copies of the coat protein and in Dec that are important for binding, as well as the structural key to the binding selectivity, its not entirely clear what general principles have been discovered. Why does phage L have a Dec protein, but P22 has none? Why is Dec binding selective at all – ie, what difference is there between Dec binding nowhere, everywhere, and in the specific location identified? Nonetheless, as a model system the structural and binding results make an interesting story that is well supported by the data and offers a foundation for continuing to pursue important questions.The manuscript could be greatly improved by correcting numerous errors, better wording, and a more concise presentation – including the Introduction, and NMR section in Results that descends into Discussion. Some examples are listed below, but others abound. A thorough read by all authors might really tighten this up.not package the genome into a preformed capsid etc. And yet there is a well-defined group in which this is, as far as I know, true – dsDNA tailed phages and herpesviruses. Why not say that?  The third sentence then says "Phages and viruses have evolved diverse strategies to strengthen their capsids, such as non-covalent binding of auxiliary "decoration" proteins." This appears to be true of the dsDNA tailed phages and herpesviruses, but not necessarily for capsids that must disassemble to deliver their genomes, such as the dsDNA polyoma/papillomaviruses . The point being to word these statements precisely so as to avoid inadvertently generalizing in ways that are simply not correct.1) Be careful to specify the scope of statement – e.g., the Abstract starts "The major coat proteins of most dsDNA tailed phages and viruses form capsids by a mechanism that includes active packaging of the dsDNA genome into a precursor procapsid, and subsequent expansion/stabilization of the capsid." By "most" the implication is that some dsDNA tailed phages and viruses do 2) Another example of a poorly chosen term "…such that some coat proteins are in hexameric conformations and some are in pentameric conformations" where strictly they are in hexavalent locations  and pentavalent location, although those are organized as pentamers. As above, if you properly specify the subset of "Viral icosahedral capsids" as pentamer-hexamer dsDNA tailed phages and herpesviruses, then your statements following aren't wrong.Similarly, in the first paragraph of the Introduction, environmental stress .31 viruses are found in the biosphere" neglects the indirect argument involved – 1030 bacteria were estimated from water samples, with 10 phage for each bacterium leading to the 1031 figure. Since phage are expected to dominate eukaryotic viruses numerically, the statement should read "1031 viruses are estimated in the biosphere". And the 2005 citation to support this  is well-preceded by Hendrix's 1999 "All the world's a phage” paper in PNAS – see the start of the Discussion."At least 103) The tight packaging of DNA "…facilitates delivery of the majority of the viral genome into the host" is supported by what evidence? The dsDNA is not a fluid released like champagne from an uncorked bottle, and in some cases only a small portion is released initially with the rest drawn out by transcription – e.g., phage T5.4) Introduction, third paragraph: The reference to chainmail should not be the 1989 Popa et al. paper, which merely indicated that the HK97 major capsid protein was involved in "wholesale covalent crosslinking", but instead the Duda 1998 Cell paper that proposed the chainmail lattice, and/or the Wikoff et al. crystal structure paper of 2000 in Science that verified it with the atomically-modeled details.5) "gpD stabilizes its capsid by binding at all three-fold symmetry axes " – first, it binds at the local and icosahedral 3-fold symmetry positions , and note that the use of "true three-fold axis" in Figure 6 and elsewhere would be better described as "icosahedral 3-fold axis". Secondly the citations are to recent structural papers that do not demonstrate that binding gpD stabilizes the capsid – that would be much earlier, Sternberg and Weisberg, 1977 or Imber et al., 1980, both in JMB. Structural data were also available much earlier, from the first lambda capsid map by Dokland and Murialdo, 1995, to the gpD crystal structure and improved capsid map by Yang et al., 2000.6) "P-loops" are not defined here, or anywhere. Indicate such domains on Figure 1—figure supplement 2.7) "The twelfth penton vertex is occupied by the portal channel…" – first, its not really a "penton vertex" since no penton occupies it; and second, while it is indeed occupied by a channel it would be more correct would be to say that its occupied by the portal complex that connects to the tail apparatus.8) The amino acid differences between phage L and P22 coat proteins are marked on Supplementary Figure 2D, but it would be useful to see them more in context by marking them (as spheres?) on Supplementary Figure 2E.9) Supplementary Figure 4 colors one of the three subunits in rainbow according to the caption – this does not appear to the case as it is cyan and purple with some abrupt point of transition. Marking the N- and C-termini, and including the fold (one subunit) may help with this figure. The main text  suggests that cyan is the N-terminal region (OB-fold) and the purple is the C-terminal region . Please clarify in the legend.10) Subsection “NMR structure of the Dec monomer has an OB-fold domain and an unfolded C terminus”, first and last paragraphs and legend to Supplementary Figure 4: Supplementary figure 5 should be Supplementary Figure 6. However, the two figures should probably be swapped as the NMR data  are referred to before the Rosetta modeling .11) Confusing as written "To further characterize the dynamics of Dec, we collected NMR 15N-relaxation data  and analyzed the dynamics of the monomeric protein in terms of the "Model-Free" formalism." Since the NMR data have already been described for the previous two pages, introducing the data collection again is weird. Perhaps it should read "To further characterize the dynamics of the monomeric Dec protein, we analyzed the NMR 15N-relaxation data  in terms of the "Model-Free" formalism."12) "We noted that coat proteins occupying different local conformations 290  contribute different residues to the binding interface ." The difference in residues contributed is not apparent in either Figure 5A nor in the video.13) Results, subsection “A structural basis for the discminatory binding of Dec to capsids”, does not include the pentamer-hexamer quasi-3-fold location, and why Dec does not bind there.14) Subsection “Decoration proteins employ a variety of binding schemes to adhere to phage capsids”, might include a reference to the herpesvirus triplex binding.15) "occupy similar spatial positions as the HK97 catalytic residues that crosslink to form the lattice "chainmail"." These residues would be better called the crosslinking residues as a catalyst by definition is not "used up" by the reaction – a third residue is proposed to act as a catalyst for the crosslinking reaction.16) "the inherent asymmetry in the Dec trimer" – this Dec asymmetry is observed in the cryoEM data where the Dec trimer is bound to the capsid at positions that depart slightly from 3-fold symmetry, and presumably the homo-trimer is flexible enough to accommodate the capsid. It is not observed in the NMR data since that is of monomeric Dec. What then is the basis for saying that this asymmetry is inherent in the Dec trimer when is it rather more certainly inherent in the capsid at the positions Dec binds to? The strongest evidence might be that Dec doesn't bind at the icosahedral (strict) 3-fold site, but then it also doesn't bind at the local 3-fold adjacent to the pentamers (and this isn't explained – see #13 above).17) The authors might consider mentioning earlier in the Introduction that gpD and Dec bind the capsids as homotrimers.18) Some figures are not easily understood, including Figure 1 where the capsomers and the T=7 organization are hard to see .Reviewer #3:In this manuscript Newcomer et al. bring three approaches to bear on the question of how phage L Dec protein discriminates between true and quasi three-fold symmetry sites. The structure of phage L coat lattice and capsid bound Dec trimers is obtained by cryo-EM, an NMR structure of a partially refolded Dec monomer is obtained, and site directed mutagenesis is performed on P22 coat protein to assess the effect on Dec binding.The techniques brought to bear are all high-resolution techniques and the cryo-EM and NMR experiments seem to be technically solid. In this reviewer's opinion the NMR studies of a partially refolded Dec monomer are something of a distraction from the focus of the manuscript.Curiously, the mutagenesis experiments were performed using bacteriophage P22 coat protein rather than phage L. This reviewer appreciates the similarity between the two phage, and also the availability of systems for genetic manipulation and production of P22 phage. While it seems likely  that binding is the same, constructing a P22 coat protein mutant containing the 4 phage L sequence alterations would seem to be straightforward and more compelling.The major reservation this reviewer has is despite the effort and high-resolution structures obtained the end result is that discriminatory binding is "largely due to the surface area of contact with the capsid protein", a vague statement which could have been made even prior to this study.There are no doubt valuable and intriguing findings in this manuscript. However, perhaps reflective of the interests of the multiple groups involved the focus drifts from discriminatory binding, to the novelty of the structure of the Dec protein itself, to unanswered questions about why some phage have decoration/stabilization proteins and some don't.[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]eLife. Your article has been reviewed favorably by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by John Kuriyan as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "The phage L capsid decoration protein has a novel OB-fold and an unusual capsid binding strategy" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This is a revised version of the manuscript by Newcomer et al. in which they describe the structure of phage L, a P22-like dsDNA phage that, unlike P22, is stabilized by decoration proteins that bind to quasi-threefold, but not icosahedral threefold positions in the capsid. Furthermore, they determine the NMR solution structure of Dec, which has an OB fold, different from e.g. the gpD decoration protein of phage lambda. Additionally, mutational analysis was used to identify the residues involved in the CP-Dec binding interaction.The authors have generally responded well to the previous reviewers' comments. Most importantly, they provide an improved map and more well refined model.Essential revisions:1) Discussion: "…decoration proteins for which atomic-resolution structures are known…" This fails to consider the Psu decoration protein of phage P4, which, like Dec, serves to stabilize the capsids and can be added to capsids extraneously, but, unlike Dec, binds across the center of hexamers . The crystal structure of Psu has been determined  and has an α-helical fold, completely different from those of T4, T5, L or lambda. Presumably Psu belongs to an even more distinct lineage of decoration proteins.2) Figure 1C is somewhat misleading, in that the color scheme suggests that the six subunits are organized around an icosahedral threefold axis. It would be more useful to color the subunits according to the quasi-symmetric location (A-G) and provide a key to understand which is which . Positions of symmetry axes (twofold?) should also be indicated.3) Figure 6: I get that the two quasi-threefold and the icosahedral threefold position differ in the angle between subunits and that this probably determines whether Dec binds or not. However, the figure is rather messy and it is difficult to see where Dec would bind to the CP. Is there a better way to represent this so that the CP-Dec interaction can be seen in the side view? Maybe a thinner slice through the structure, depth cueing or something like that?4) Furthermore, the table in Figure 6, which lists differences in distance between residue E81, does not convey the point made in the text  that the icosahedral threefold is the flattest, the Dec binding quasi-threefold is intermediate, and the pentamer-proximal position is the most angled. One might have presumed that the most angled positions that represent a "weak point in many capsids" would be the one where Dec binds. There does not seem to be much discussion of this interesting point, which may point to Dec's function – perhaps not to stabilize capsids after all?5) Also, in Figure 6, the arc with the denotation 54.2Å is confusing. What is this the distance between? It seems to indicate an angle rather than a distance. Was it meant to be 54.2°? It might be helpful to list these angles differences between all three types of trimers.6) I find it a really missed opportunity to demonstrate the claimed quality of the maps by not showing some region with excellent side-chain density with the fold model fit into it – not in the manuscript, supplementary figures or videos.7) This rebuttal statement "We have de-emphasized the notion of discriminatory binding in our title and throughout the text" does not seem to be accurate. The Abstract states: "The Dec protein from the P22-like phage L has a highly unusual binding strategy that precisely distinguishes between nearly identical three-fold and quasi-three-fold sites of the icosahedral capsid." and "Here, we investigate how Dec is able to distinguish subtly different capsid binding sites with high specificity." This aspect has rather more prominence than claimed. [Editors’ note: the author responses to the first round of peer review follow.]All reviewers felt that it is of interest to understand discrimination of QE locations in capsids. One reason might be to understand immune recognition. Another might be to be able to direct assembly toward different T numbers. Unfortunately, the reviewers were not convinced that the paper gave insights into the structural basis for this in phage L, and whether this might be generalized to other viruses. Given that eLife does not make major revision decisions where substantial new work would be required, the only option was to reject the current submission. However, this does not preclude the future resubmission of a revised manuscript that fully addresses the concerns raised in this review.Thank you for the suggestions and for the opportunity to make the text more generalizable to a broad audience. We have addressed these points in the Introduction and Discussion sections to include how this is generalizable to other viruses, nanomedicine, general assembly and stability mechanisms and also of great utility in nanomaterials design. We also emphasized the point that an OB-fold is a novel domain for decoration proteins and this is a fundamentally important finding within this work. We have indicated that the decoration proteins discussed specifically in our manuscript bind only after maturation, so they cannot affect T number.Reviewer #1:[…]1) The stated resolution of the cryo-EM map is ambiguous and misleading. The authors report a 4.2 Å overall resolution of the icosahedral reconstruction . But from Figure 1C, one can clearly tell the map resolution for the Dec protein part is probably around 8~15 Å, much worse than 4.2 Å. Also, the half-map FSC is measuring self-agreement between two maps rather than actual resolution. Therefore the authors need to measure the Model:Map FSC for the capsid proteins and Dec separately, and include those numbers in the paper.We have run map validation procedures using Phenix and have listed this information in the Materials and Methods section. By applying the autosharpening routine, the display of the map is much improved, and the new figures accurately reflect the high resolution. Indeed, this capsid reconstruction has varying resolution at different radii. We have tried to further emphasize this point where we talk about the local resolution assessment . Selected statistics from the validation report are now shown in Table 1.2) The features in this cryo-EM map do not look like a typical 4.2 Å map. For example, one usually sees side chain densities on a helix and β-strand separation at this resolution. But these were not seen in the map provided by the authors. Therefore, it is either the authors over-estimated the cryo-EM resolution, or the map has not been properly sharpened. With the map quality as seen, it is hard to believe the detailed amino acid site analysis presented in Figure 5. This also suggests that some of the mutagenesis results can simply be explained by an inaccurate atomic model.We appreciate the point made by the reviewer and we acknowledge we were in error in supplying the raw data for review. We have now supplied a sharpened map, where the highresolution quality is easier to visualize.After using the autosharpening routine within Phenix, the display of the map is much improved, and the new figures accurately reflect the high resolution. The detailed side chain information that guided the mutagenesis experimentation is well supported by three independent points:1) The phage L coat protein density is of sufficient quality to determine a complete model de novo.https://www.ncbi.nlm.nih.gov/pubmed/28270620).2) Phage L coat protein has 99.6% identity to P22 coat protein. The majority of our phage L model is within 1-1.5 Å RMSD from the high-resolution models derived from P22 data  Our NMR data is derived independently. The NMR data accurately describes the N-terminal portion of Dec protein (residues 1-77), and docks in very well with the cryo-EM derived models. While we agree that the C-terminal β-helix is much lower resolution in our cryo-EM map, only the N-terminal OB fold of the Dec protein as docked into the trimer is necessary for designing/interpreting site-directed mutagenesis studies.Therefore, a hypothesis regarding detailed side chain information where the coat protein contacts Dec is a perfectly reasonable one to make and to test. The experimental evidence from the mutagenesis data aligns with our predictions and gives additional strength to the atomic models of coat and Dec. In fact, ours is one of very few papers that experimentally tests decoration protein binding to capsids via mutagenesis, and we believe this significantly adds to the current understanding within the field. We have made it a point to more clearly state the value of this experiment throughout the text.3) Both the capsid protein and Dec models sent from the authors have poor protein geometries, meaning they were poorly refined. Both models even have cis-peptides in the structure, which is unacceptable. The authors need to put a table in the main text listing protein geometries  of the deposited cryo-EM models and maps, and those statistics need to reach the common standard of the field.We have used Phenix to perform real-space refinement within the sharpened density and have improved models. Furthermore, we have deposited the validation reports along with our models to the EMDB. Selected statistics from the validation report are now shown in Table 1. The full validation reports for the capsid protein are available to anyone who wants access under the accession number EMD-9392. The NMR structure has been deposited to the PDB and validation reports on that structure are also publically available under the accession number 6E3C.4) One of the most interesting points in the paper involves the Dec selectivity on the Phage L capsid. But why were the mutagenesis experiments done using Phage P22, which doesn't have Dec?This is a result of two considerations:1) Using P22 drastically increased efficiency and reduced the cost of the experiments. Since P22 is a well-established model system, we had access to clear plaque mutants, amber mutants, non-sense tRNA suppressors, cloned plasmids, a variety of host strains etc. We would need to establish a complete genetic system in phage L in order to do these experiments. Since previous work showed that P22 and phage L capsids are virtually identical , we decided there was no justification to spend the time and money on the creation of an entirely new Phage L genetic system.2) P22 is a well-established model system that is used by many laboratories for molecular display and nano-materials design. Dec is often used in these experiments with P22. Therefore, it is very useful to have the mutant binding data using P22 particles. See a large body of work by Trevor Douglas’ group (>30 publications) as well as nanomaterials work from other groups.5) The structural basis for the selective binding of Dec to capsids was poorly discussed. The MD-based simulations are not helpful in elucidating the mechanism. First, the authors need to understand that the hexons in the capsid do not necessarily have C6 symmetry. This is true from the model sent by the authors, and this means the "quasi-three-fold" and "true three-fold" may have different surface interfaces to Dec, which is the simplest explanation for Dec selectivity. With a 4.2 Å map, the author should be able to compare the surface using real cryo-EM map/models, rather than some MD simulations.Unfortunately, MD was the only way to answer this question, as there was no significant cryo-EM density of Dec at the true icosahedral three-fold site. Therefore, the only way we could possible examine how Dec might interact with the surface was computationally. We couldn’t use “real” cryo-EM map/models without evidence of real density.In light of this reviewer’s concern over the use of computational methodology, we have removed this result from the current work.Reviewer #2:The manuscript "How phage L capsid decoration protein distinguishes between nearly identical binding sites on an icosahedral virion" by Newcomer et al. looks at the binding of Dec on the capsid exterior surface in its host phage L, which varies according to position on the capsid surface. In addition, Dec binds to phage P22 due to 99.6% identity in coat protein sequence with that of phage L, although P22 doesn't encode its own Dec homolog. Capsid structures determined by high-resolution cryo-electron microscopy are combined with an NMR solution structure of monomeric Dec, MD simulations, and mutations across the P22 core and Dec interface to reveal the selectivity of Dec binding. Although this is a rather specific example from one of many similar phages, it nonetheless reflects a more general question of the fidelity of protein-protein binding that can also be found in other cellular contexts and for regulating function. Thus, it's a useful illustration of a general process that is readily studied in the tractable context of high-symmetry phage particles that are easily expressed and purified. However, while identifying several residues across multiple copies of the coat protein and in Dec that are important for binding, as well as the structural key to the binding selectivity, its not entirely clear what general principles have been discovered. Why does phage L have a Dec protein, but P22 has none? Why is Dec binding selective at all – ie, what difference is there between Dec binding nowhere, everywhere, and in the specific location identified? Nonetheless, as a model system the structural and binding results make an interesting story that is well supported by the data and offers a foundation for continuing to pursue important questions.The manuscript could be greatly improved by correcting numerous errors, better wording, and a more concise presentation – including the Introduction, and NMR section in Results that descends into Discussion. Some examples are listed below, but others abound. A thorough read by all authors might really tighten this up.Thank you for finding value in the story and the data. We have significantly improved the Introduction and Discussion and have removed extraneous detail in the NMR section. We hope this makes the manuscript easier to read. We also think it’s interesting that phage L encodes a Dec protein while phage P22 does not. Given that the phage P22 capsid is very stable and its coat protein has only 4 of 430 a.a. different from phage L, it is puzzling that the latter phage would make an extra stabilizing protein given the pressures on viruses to minimize their genomes for efficient packaging. One possible solution to this paradox is if Dec had a second function besides capsid stabilization such as cell recognition. We have evidence that Dec might bind to target cells, presumably through carbohydrate moieties emanating from the target cell surfaces. From a structural point of view, both the OB-fold part of Dec and the C-terminal β-helix could be carbohydrate binding motifs. Because our data at this point is preliminary and pertains to a different scope – capsid-binding of Dec, we did not include it in the present manuscript. If Dec indeed turns out to have a second targetcell binding function, the selectivity of Dec binding could serve a structural role – namely different surface patterning on the icosahedral lattice, that could be used for target-cell binding selectively.1) Be careful to specify the scope of statement – eg, the Abstract starts "The major coat proteins of most dsDNA tailed phages and viruses form capsids by a mechanism that includes active packaging of the dsDNA genome into a precursor procapsid, and subsequent expansion/stabilization of the capsid." By "most" the implication is that some dsDNA tailed phages and viruses do not package the genome into a preformed capsid etc. And yet there is a well-defined group in which this is, as far as I know, true – dsDNA tailed phages and herpesviruses. Why not say that?  The third sentence then says "Phages and viruses have evolved diverse strategies to strengthen their capsids, such as non-covalent binding of auxiliary "decoration" proteins." This appears to be true of the dsDNA tailed phages and herpesviruses, but not necessarily for capsids that must disassemble to deliver their genomes, such as the dsDNA polyoma/papillomaviruses . The point being to word these statements precisely so as to avoid inadvertently generalizing in ways that are simply not correct.Thank you for the suggestion. We have addressed these points by specifically stating “dsDNA phages and herpesviruses” to be explicit.2) Another example of a poorly chosen term "…such that some coat proteins are in hexameric conformations and some are in pentameric conformations" where strictly they are in hexavalent locations  and pentavalent location, although those are organized as pentamers. As above, if you properly specify the subset of "Viral icosahedral capsids" as pentamer-hexamer dsDNA tailed phages and herpesviruses, then your statements following aren't wrong.We changed our text to be more explicit.Similarly, in the first paragraph of the Introduction, environmental stress .We changed our text to add the point regarding dsDNA packaging.31 viruses are found in the biosphere" neglects the indirect argument involved – 1030 bacteria were estimated from water samples, with 10 phage for each bacterium leading to the 1031 figure. Since phage are expected to dominate eukaryotic viruses numerically, the statement should read "1031 viruses are estimated in the biosphere". And the 2005 citation to support this  is well-preceded by Hendrix's 1999 "All the world's a phage” paper in PNAS – see the start of the Discussion."At least 10Unfortunately, we have to disagree with this reviewer on this point. The subject of the Hendrix 1999 paper is evolutionary relationships that align DNA sequences and protein sequences to infer relatedness among diverse phages. There are no data presented in that work that serves as an estimate for the total number of phages in the biosphere.The Hendrix paper does however reference a 1989 paper from Bergh et al. that estimates the abundances of viruses in aquatic environments. In addition, there was an updated report in 2000 from Wommack et al. We have replaced the Bamford reference with these two papers to provide references that precede 2005.3) The tight packaging of DNA "…facilitates delivery of the majority of the viral genome into the host." Is supported by what evidence? The dsDNA is not a fluid released like champagne from an uncorked bottle, and in some cases only a small portion is released initially with the rest drawn out by transcription – e.g., phage T5.There is an enormous body of evidence to support the idea that the pressure inside the capsid is sufficient to eject some or in many cases most of the dsDNA chromosome for a variety of phages. We have added these references to the paper and clarified the statement that this phenomenon relates to “many phages and herpesviruses” so as not to imply that this is true for T5:Some seminal original research papers on pressure and ejection of phages and Herpesviruses:1) Evilevitch, 20182) Kellermeyer et al., 20183) Bauer et al., 20134) Bauer et al., 20155) Jin et al. 2015 Virology:https://www.ncbi.nlm.nih.gov/pubmed/?term=jin+parent+gelbart6) Gelbart and Knobler, 20097) Grayson et al., 2006 Virology: https://www.ncbi.nlm.nih.gov/pubmed/164693468) Kindt et al., 2001Some review papers:1) Molineux and Panja 2013 Nat Rev Microbiol:https://www.ncbi.nlm.nih.gov/pubmed/233857862) Roos et al., 2007 Cell Mol Life Sci: https://www.ncbi.nlm.nih.gov/pubmed/174406804) Introduction, third paragraph: The reference to chainmail should not be the 1989 Popa et al. paper, which merely indicated that the HK97 major capsid protein was involved in "wholesale covalent crosslinking", but instead the Duda 1998 Cell paper that proposed the chainmail lattice, and/or the Wikoff et al. crystal structure paper of 2000 in Science that verified it with the atomically-modeled details.Thank you for the suggestion. We have added the Duda and Wikoff references to the end of the sentence, but also kept the Popa reference where we state that the capsid is stabilized by crosslinks as this was the first paper to demonstrate covalent crosslinking phenomenon.5) "gpD stabilizes its capsid by binding at all three-fold symmetry axes " – first, it binds at the local and icosahedral 3-fold symmetry positions , and note that the use of "true three-fold axis" in Figure 6 and elsewhere would be better described as "icosahedral 3-fold axis". Secondly the citations are to recent structural papers that do not demonstrate that binding gpD stabilizes the capsid – that would be much earlier, Sternberg and Weisberg, 1977, or Imber et al., 1980, both in JMB. Structural data were also available much earlier, from the first lambda capsid map by Dokland and Murialdo, 1995, to the gpD crystal structure and improved capsid map by Yang et al., 2000.Thank you for the suggestion. We have replaced all instances of “true three-fold” with “icosahedral three-fold”.Thank you also for the suggestions of additional references. It was not our intention to leave out critical findings, and we have happily added the Sternberg and Weisberg, Imber, and Dokland Murialdo citations in the Introduction where we first describe gpD.6) "P-loops" are not defined here, or anywhere. Indicate such domains on Figure 1—figure supplement 2.Label added.7) "The twelfth penton vertex is occupied by the portal channel…" – first, its not really a "penton vertex" since no penton occupies it, how about dropping "penton"; and second, while it is indeed occupied by a channel it would be more correct would be to say that its occupied by the portal complex that connects to the tail apparatus.The word “penton” has been removed. “Channel” has been changed to “complex”.8) The amino acid differences between phage L and P22 coat proteins are marked on Supplementary Figure 2D, but it would be useful to see them more in context by marking them (as spheres?) on Supplementary Figure 2E.It proved very difficult to show this well in a 2D view, so instead we made a video showing the location of these differences surrounding a Dec binding site at a quasi three-fold axis (new Video 1).9) Supplementary Figure 4 colors one of the three subunits in rainbow according to the caption – this does not appear to the case as it is cyan and purple with some abrupt point of transition. Marking the N- and C-termini, and including the fold (one subunit) may help with this figure. The main text  suggests that cyan is the N-terminal region (OB-fold) and the purple is the C-terminal region . Please clarify in the legend.This point is no longer relevant as we have removed this figure from the manuscript.10) Subsection “NMR structure of the Dec monomer has an OB-fold domain and an unfolded C terminus”, first and last paragraphs and legend to Supplementary Figure 4: Supplementary Figure 5 should be Supplementary Figure 6. However, the two figures should probably be swapped as the NMR data  are referred to before the Rosetta modeling .In response to several comments by the reviewers, we have reordered the text and the corresponding figures to match in several places.11) Confusing as written "To further characterize the dynamics of Dec, we collected NMR 15N-relaxation data  and analyzed the dynamics of the monomeric protein in terms of the "Model-Free" formalism." Since the NMR data have already been described for the previous two pages, introducing the data collection again is weird. Perhaps it should read "To further characterize the dynamics of the monomeric Dec protein, we analyzed the NMR 15N-relaxation data  in terms of the "Model-Free" formalism."Thank you for the suggestion. Fixed.12) "We noted that coat proteins occupying different local conformations 290  contribute different residues to the binding interface ." The difference in residues contributed is not apparent in either Figure 5A nor in the video.We have adjusted the figure, the videos and the legend to make this more clear.13) Results, subsection “A structural basis for the discminatory binding of Dec to capsids”, does not include the pentamer-hexamer quasi-3-fold location, and why Dec does not bind there.We have de-emphasized the notion of discriminatory binding in our title and throughout the text.14) Subsection “Decoration proteins employ a variety of binding schemes to adhere to phage capsids”, might include a reference to the herpesvirus triplex binding.We added two references: an original structure paper that first shows the triplexes  and a review article .15) "occupy similar spatial positions as the HK97 catalytic residues that crosslink to form the lattice "chainmail"." These residues would be better called the crosslinking residues as a catalyst by definition is not "used up" by the reaction – a third residue is proposed to act as a catalyst for the crosslinking reaction.We have changed “catalytic” to “crosslinking”.16) "the inherent asymmetry in the Dec trimer" – this Dec asymmetry is observed in the cryoEM data where the Dec trimer is bound to the capsid at positions that depart slightly from 3-fold symmetry, and presumably the homo-trimer is flexible enough to accommodate the capsid. It is not observed in the NMR data since that is of monomeric Dec. What then is the basis for saying that this asymmetry is inherent in the Dec trimer when is it rather more certainly inherent in the capsid at the positions Dec binds to? The strongest evidence might be that Dec doesn't bind at the icosahedral (strict) 3-fold site, but then it also doesn't bind at the local 3-fold adjacent to the pentamers (and this isn't explained – see #13 above).We have removed this statement.17) The authors might consider mentioning earlier in the Introduction that gpD and Dec bind the capsids as homotrimers.We have added this detail to the Introduction.18) Some figures are not easily understood, including Figure 1 where the capsomers and the T=7 organization are hard to see .We have replaced Figure 1A with a cartoon schematic that better shows T = 7 organization. Supplementary Figure 2 is now removed in the revised text.Reviewer #3:In this manuscript Newcomer et al. bring three approaches to bear on the question of how phage L Dec protein discriminates between true and quasi three-fold symmetry sites. The structure of phage L coat lattice and capsid bound Dec trimers is obtained by cryo-EM, an NMR structure of a partially refolded Dec monomer is obtained, and site directed mutagenesis is performed on P22 coat protein to assess the effect on Dec binding.The techniques brought to bear are all high-resolution techniques and the cryo-EM and NMR experiments seem to be technically solid. In this reviewer's opinion the NMR studies of a partially refolded Dec monomer are something of a distraction from the focus of the manuscript.We reworked the text surrounding the NMR data to highlight the important parts, but to decrease on the extraneous details. We have recent unpublished data that freshly purified Dec trimer slowly dissociates to monomers over a period of about a week in solution. Thus the Dec trimer seems to be a metastable kinetically trapped state in solution. Whether the capsids or other intracellular factors stabilize the Dec trimer is an open question, but our results suggest that the monomeric form of Dec is a mechanistically important species. We are currently investigating the mechanism of Dec trimer formation and dissociation. Lastly, the NMR data for the OB-fold was the basis for building the trimer into the cryo-EM density map as well as guiding site-directed mutagenesis, and thus us critical to the work.Curiously, the mutagenesis experiments were performed using bacteriophage P22 coat protein rather than phage L. This reviewer appreciates the similarity between the two phage, and also the availability of systems for genetic manipulation and production of P22 phage. While it seems likely  that binding is the same, constructing a P22 coat protein mutant containing the 4 phage L sequence alterations would seem to be straightforward and more compelling.We chose not to make these substitutions as none of them are involved in Dec binding, or near the binding sites. If any were involved in this binding interface we would have expected the binding properties of Dec  to be affected when comparing binding to P22 or L. Additionally, each of the amino acids that are changed in L are found in some P22 stocks as phenotypically silent mutations , suggesting that these minor differences play no role in the way the phage capsids assemble or function. We have added this description to the text.The major reservation this reviewer has is despite the effort and high-resolution structures obtained the end result is that discriminatory binding is "largely due to the surface area of contact with the capsid protein", a vague statement which could have been made even prior to this study.There are no doubt valuable and intriguing findings in this manuscript. However, perhaps reflective of the interests of the multiple groups involved the focus drifts from discriminatory binding, to the novelty of the structure of the Dec protein itself, to unanswered questions about why some phage have decoration/stabilization proteins and some don't.We have emphasized the novelty of the Dec OB-fold, as well as the discovery of which residues contribute to capsid binding. These observations would not have been possible without the high-resolution work contained here.We have de-emphasized the discussion regarding selectivity of binding in some places, although we have added a new figure  that shows how the coat protein structures change at the different sites to make this more obvious.[Editors' note: the author responses to the re-review follow.]Essential revisions:1) Discussion: "…decoration proteins for which atomic-resolution structures are known…" This fails to consider the Psu decoration protein of phage P4, which, like Dec, serves to stabilize the capsids and can be added to capsids extraneously, but, unlike Dec, binds across the center of hexamers . The crystal structure of Psu has been determined  and has an α-helical fold, completely different from those of T4, T5, L or lambda. Presumably Psu belongs to an even more distinct lineage of decoration proteins.Thank you for this addition. We have added these references to the Discussion  and included a mention that Psu is from another category of decoration proteins.2) Figure 1C is somewhat misleading, in that the color scheme suggests that the six subunits are organized around an icosahedral threefold axis. It would be more useful to color the subunits according to the quasi-symmetric location (A-G) and provide a key to understand which is which . Positions of symmetry axes (twofold?) should also be indicated.We have adjusted the figure according to the color scheme recommended by the reviewers and added an enlarged area to show the fit of the map and the high resolution allowing us to view side chains. The figure legend has been edited accordingly.3) Figure 6: I get that the two quasi-threefold and the icosahedral threefold position differ in the angle between subunits and that this probably determines whether Dec binds or not. However, the figure is rather messy and it is difficult to see where Dec would bind to the CP. Is there a better way to represent this so that the CP-Dec interaction can be seen in the side view? Maybe a thinner slice through the structure, depth cueing or something like that?We have updated the figure to show additional views including an overlay, as well as two individual views of the quasi three-fold between hexamers (light blue) as well as the quasi three-fold site between hexamers and pentamers (dark blue). We also included the Dec density (lavender) in this new figure to emphasize the differences between these two quasi equivalent environments.4) Furthermore, the table in Figure 6, which lists differences in distance between residue E81, does not convey the point made in the text  that the icosahedral threefold is the flattest, the Dec binding quasi-threefold is intermediate, and the pentamer-proximal position is the most angled.We have expanded the Discussion in a few places to address the point regarding E81 positioning at the various quasi-equivalent locations more clearly (see subsection “Decoration proteins employ a variety of binding schemes to adhere to phage capsids”).One might have presumed that the most angled positions that represent a "weak point in many capsids" would be the one where Dec binds. There does not seem to be much discussion of this interesting point, which may point to Dec's function – perhaps not to stabilize capsids after all?We have revised this statement to more clearly explain that Dec does bind to the capsids where they suffer from AFM damage and are thus a weak point:“Biophysical measurements have shown that Dec plays a much larger role in stabilizing dsDNAcontaining heads compared to empty capsids, and atomic force microscopy experiments showed Dec indeed binds capsids at positions most prone to mechanical damage (i.e. quasi three-fold sites between hexons) ”5) Also, in Figure 6, the arc with the denotation 54.2Å is confusing. What is this the distance between? It seems to indicate an angle rather than a distance. Was it meant to be 54.2°? It might be helpful to list these angles differences between all three types of trimers.The intention of the distance measurement was to emphasize the large difference in curvature. However, we appreciate that this was confusing and have since removed this label in the new figure. We hope that the new figure is clearer and that the presence of the Dec density sufficiently emphasizes the distance without the need for listing additional direct measurements. We have also updated the figure legend accordingly.6) I find it a really missed opportunity to demonstrate the claimed quality of the maps by not showing some region with excellent side-chain density with the fold model fit into it – not in the manuscript, supplementary figures or videos.We added an enlarged area view to Figure 1 showing the fit of side chains into the cryo-EM density map of the capsid protein spine helix.7) This rebuttal statement "We have de-emphasized the notion of discriminatory binding in our title and throughout the text" does not seem to be accurate. The Abstract states: "The Dec protein from the P22-like phage L has a highly unusual binding strategy that precisely distinguishes between nearly identical three-fold and quasi-three-fold sites of the icosahedral capsid." and "Here, we investigate how Dec is able to distinguish subtly different capsid binding sites with high specificity." This aspect has rather more prominence than claimed.We have deleted the word “precisely” in the Abstract. We also deleted the statement “Here, we investigate how Dec is able to distinguish subtly different capsid binding sites with high specificity.”Additionally, we have revised the next sentence to read: “We report the structure of phage L Dec protein, which has a novel fold for a decoration protein and propose an explanation for how Dec may be able to bind to subtly different capsid binding sites.”


Carbonic anhydrases (CAs) are ubiquitous metallo-enzymes that catalyse the reversible hydration of carbon dioxide to bicarbonate and proton. In humans there are 15 isoforms among which only 12 are catalytically active. Since active human (h) CAs show different efficiency, the understanding of the molecular determinants affecting it is a matter of debate. Here we investigated, by a site-specific mutagenesis approach, residues modulating the catalytic features of one of the least investigated cytosolic isoform, i.e. hCA XIII. Results showed that residues assisting the formation of an ordered solvent network within the catalytic site as well as those forming a histidine cluster on the protein surface are important to guarantee an efficient proton transfer. Since the binding of the latter ion to the zinc is rather labile, it is replaced by a water molecule leading to the catalytically inactive form of the enzyme (2+-bound hydroxide species through a proton transfer reaction from the zinc-coordinated water molecule to the bulk solvent (Equation (2))In the first step, the ZnKcat/KM values of 1.5 × 108 M−1 s−1 and 8.3 × 107 M−1 s−1 for hCA II and hCA VII, respectively), hCA I and hCA XIII possess medium efficiency (for hCA I Kcat/KM = 5.0 × 107 M−1 s−1 and for hCA XIII Kcat/KM = 1.1 × 107 M−1 s−1), while hCA III acts as a very poor catalyst (Kcat/KM = 3.0 × 105 M−1 s−1)2+-bound solvent molecule to His64  has been used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes as buffer (pH 7.4). About 20 mM NaClO4 were also added to the assay system for maintaining constant ionic strength. The initial rates of the CA-catalysed CO2 hydration reaction were followed for a period of 10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. The uncatalysed rates were determined in the same manner and subtracted from the total observed rates, as reported earlierAll measurements were done according to Khalifah’s stopped flow method,,,2 hydration activity of the purified mutants was determined by a stopped-flow CO2 hydration assay and compared with activity of the wild-type hCA XIII. Results, reported in In order to design suitable hCA XIII variants, a careful comparison between the crystallographic structures of hCA XIIIThe aim of this work was to identify hCA XIII residues that determine the lower catalytic efficiency of this isoform with respect to the most efficient hCA II. Upon structural comparison, diverging residues involved in the formation of an ordered water network within the catalytic site, of a histidine cluster on the protein surface, or located in the proximity of the substrate binding pocket were replaced with those corresponding to hCA II by a site-specific mutagenesis approach. Although none of the designed variants fully restored hCA II activity, which is driven by a multifactorial basis, most of the single mutations were able to improve the enzymatic activity. Overall, our results add another piece in the puzzle of hCA catalytic mechanism highlighting that residues assisting the formation of the ordered solvent network are important to guarantee an efficient proton transfer. Moreover, also the histidine cluster represents a structural feature that contributes to the catalytic rate of this superfamily of enzymes.


First Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms (DMM), helping early-career researchers promote themselves alongside their papers. Scott Collum is first author on ‘ Scott CollumHow would you explain the main findings of your paper to non-scientific family and friends?Combined pulmonary fibrosis and emphysema (CPFE) is a disease that involves both destruction of the lung tissue (emphysema) and inappropriate scarring of the lung tissue (fibrosis). These changes, along with an increase of the blood pressure in the lungs known as pulmonary hypertension, are a deadly combination with no drug treatment available. In tissues from patients with CPFE and pulmonary hypertension, we discovered that the adenosine receptor ADORA2B was elevated in sick lungs compared to healthy lungs. This increased signaling also led to an increase in HAS3 protein, which makes hyaluronan, a molecule that is part of the scaffolding of the lung. In our mouse model of CPFE and pulmonary hypertension, in which adenosine accumulates, we saw similar changes in HAS3 levels. Using 4-methylumbeliferone (4MU), which reduces hyaluronan, we improved the lungs in our mouse model. 4MU is a US Food and Drug Administration (FDA)-approved drug not previously used in the investigation of CPFE and pulmonary hypertension. This could lead to exciting possibilities for future studies and treatment.What are the potential implications of these results for your field of research?My research shows a novel pathway involved in this complicated and hard-to-model chronic lung disease. The use of this unique model of CPFE allowed us to determine the role of adenosine signaling in the development of disease. Of particular interest is the discovery of HAS3-expressing macrophages. Hyaluronan synthases have been shown to play a role in chronic lung diseases in previous work by our lab and others. Macrophages are also an important driver in many mechanisms of chronic lung diseases. It has not previously been shown that macrophages could play an important role in the accumulation of hyaluronan in the damaged areas of the lung. The finding that 4MU treatment will reduce this activity of macrophages and lead to reduced features of CPFE in our model allows for the continued investigation of hyaluronan as a driver of this disease and offers a potential treatment for patients with CPFE.“The finding that 4MU treatment will reduce […] features of CPFE in our model […] offers a potential treatment for patients with CPFE.”What are the main advantages and drawbacks of the model system you have used as it relates to the disease you are investigating?Ada−/− mouse model recapitulates the features of CPFE with secondary pulmonary hypertension quite well, with readily apparent fibrotic deposition and airway enlargement by histology and increased pulmonary vascular pressures. Another advantage is that ADA is supplemented for these mice during development in the form of PEG-ADA. This allows for reduction of ADA at a controlled time and gradual accumulation of adenosine in a more chronic manner. Unfortunately, the supplement of PEG-ADA must be used in maintenance of the colony and for several months for each experimental mouse. In addition, important environmental stimuli, such as cigarette smoke or environmental particulate matter exposure, that are associated with CPFE do not form part of the pathophysiological response in our model.The What has surprised you the most while conducting your research?I was most surprised by how well this new model recapitulated the key features of CPFE, both the obliteration of emphysema and the fibrotic deposition with secondary pulmonary hypertension. The model even recapitulates the novel findings of HAS3-positive macrophages.Describe what you think is the most significant challenge impacting your research at this time and how will this be addressed over the next 10 years?The greatest challenges in the chronic lung disease field are the relatively small number of animal models for each disease and the difficulty in obtaining patient tissues. As this paper and others in the field have shown, many people are working to develop new models of chronic lung disease that recapitulate more accurately the symptoms and mechanisms involved in the human disease progression. In addition, collaborative efforts have begun to allow for greater access to human samples, which greatly help the translation of exciting findings in the novel animal models.What changes do you think could improve the professional lives of early-career scientists?I believe that two separate initiatives would improve early-career scientist training. First, an increased focus on funding predoctoral fellowships would allow for an important first step in gaining independence in an academic setting and would propel this career path forward. Second, a widespread collaborative training program with industry would allow for a clear career path for those that transition out of academia. Together these would reduce the uncertainty that many early-career scientists feel.“I believe that two separate initiatives would improve early-career scientist training. First, an increased focus on funding predoctoral fellowships […] Second, a widespread collaborative training program with industry.”What's next for you?I am finishing a research project on RNA processing in pulmonary hypertension that may help to explain how hyaluronan synthases are regulated in disease. I am preparing to defend my thesis on this project. I am also searching for postdoctoral positions with a continued focus on translational research.