When we eat a meal, bile salts in the intestine help the body to absorb molecules of fat. Certain bacteria that cause food poisoning in humans, such as Vibrio parahaemolyticus, can also detect bile salts and use them to sense that they have reached the intestine of a suitable host. The bacteria then produce toxins that inflame the intestine; this can result in days of diarrhea. However, it was not clear how the bacteria detect the bile salt signal and how this triggers them to produce the toxins.

Li, Rivera-Cancel et al. now discover that not one, but two, genes are required for this process. The genes encode two proteins, called VtrA and VtrC, that interact to form protein ‘complex’ on the surface of the membrane that surrounds the bacterial cell. The two proteins create a barrel-like structure that can bind to bile salts and trigger the cell to produce the toxins.

Future experiments will aim to understand how the binding of bile salts to this protein complex causes an increase toxin production. A future challenge is to find out how other disease-causing bacteria sense environmental cues to produce toxins. With this knowledge, researchers might be able to design new drugs that could prevent the production of toxins to relieve symptoms of food poisoning and other illnesses.

Bile is an important component of the human gastrointestinal tract that plays a major role in the emulsification and solubilization of lipids (Begley et al., 2005). Its main components are bile salts, cholesterol, and phospholipids that together possess antimicrobial activities, challenging the survival and colonization of both pathogenic and commensal bacteria (Begley et al., 2005). There is increasing evidence that enteric bacterial pathogens can utilize bile as a signaling cue to regulate gene expression profiles during host infection. For example, Salmonella typhimurium senses bile to repress the genes involved in invasion when growing in the intestinal lumen and induce their expression to promote invasion upon the penetration of the mucosal layer wherein the concentration of bile is decreased (Prouty and Gunn, 2000; Prouty et al., 2004). In Shigella spp., bile salts increase the pathogen's adherence and invasion of epithelial cells (Pope et al., 1995). Bile also has important effects on the pathogenicity of Vibrio species, as observed in multiple studies with pathogenic strains of V. cholerae and V. parahaemolyticus (Gupta and Chowdhury, 1997; Schuhmacher and Klose, 1999; Krukonis and DiRita, 2003; Faruque et al., 1998). V. cholerae produces two major virulence factors during infection, cholera toxin (CT) and toxin-coregulated pilus (TCP), and these factors are repressed by ToxT in the presence of bile (Gupta and Chowdhury, 1997; Schuhmacher and Klose, 1999; Krukonis and DiRita, 2003; Faruque et al., 1998). However, it was also reported that bile can activate the production of CT independent of ToxT (Hung and Mekalanos, 2005). Non-O1/non-O139 V. cholerae strains that do not encode CT and TCP can cause gastroenteritis and utilize bile to activate a pathogenic type III secretion system (Dziejman et al., 2005; Chaand et al., 2015; Alam et al., 2010). Similarly, V. parahaemolyticus type III secretion system 2 (T3SS2) is induced specifically by bile salts during infection, resulting in acute gastroenteritis (Gotoh et al., 2010). Despite these important discoveries, the mechanism of sensing bile salts by pathogenic bacteria remains unknown. To investigate this mystery, we used V. parahaemolyticus as a model to elucidate how Vibrio spp. sense bile salts as a signal to regulate the expression of virulence genes.

V. parahaemolyticus is a globally-spread, Gram-negative, halophilic bacterial pathogen that inhabits marine and estuarine environments and is the world’s leading cause of acute gastroenteritis due to the consumption of raw or undercooked seafood (Broberg et al., 2011; Zhang and Orth, 2013). During recent years, rising temperatures in the ocean has contributed to this pathogen’s worldwide dissemination (Nair et al., 2007; Velazquez-Roman et al., 2013; McLaughlin et al., 2005; O'Boyle and Boyd, 2014; Daniels et al., 2000). V. parahaemolyticus is also the causative agent of the devastating shrimp disease Acute Hepatopancreatic Necrosis Disease (AHPND) (Tran et al., 2013; Lee et al., 2015).

Over the last decade, enormous progress has been made to elucidate virulence factors used by this pathogen and tools are available to study virulence mechanisms at the genetic and biochemical levels (de Souza Santos et al., 2015). As part of its virulence repertoire, V. parahaemolyticus encodes two T3SSs: the cytotoxic T3SS1 and the enterotoxic T3SS2. T3SSs encode needle-like secretion apparatuses used to deliver bacterial effector proteins, called Vops, which allow V. parahaemolyticus to invade and kill infected host cells (Broberg et al., 2011; Makino et al., 2003; Galán and Wolf-Watz, 2006). Each T3SS delivers a specific set of Vops into a host cell (Broberg et al., 2011). For example, the T3SS1 exclusively secretes VopQ, VopR, VPA0450 and VopS and uses the effectors to orchestrate a multifaceted host cell death. This system is found in all strains of V. parahaemolyticus, and therefore is proposed to be important for bacterial survival in the environment (Broberg et al., 2011).

T3SS2 is present in clinical isolates of V. parahaemolyticus and is a key bile salt-induced virulence system that causes enterotoxicity and symptoms associated with gastroenteritis during infection (Broberg et al., 2011; Park et al., 2004; Hiyoshi et al., 2010; Ritchie et al., 2012). Bile salts not only induce Vops, such as VopA and VopC, but also the needle-like secretion apparatus, including components such as T3SS2 translocon VopD2 (Broberg et al., 2011; Zhang and Orth, 2013; Trosky et al., 2004). In V. parahaemolyticus, the activation of T3SS2 by bile salts is regulated by two transmembrane ToxR-like transcription factors, VtrA (VPA1332) and VtrB (VPA1348) (Gotoh et al., 2010; Kodama et al., 2010). Homologues of VtrA and VtrB, named VttRA and VttRB respectively, have been identified in T3SS-containing non-O1/non-O139 V. cholerae strains and function in a similar way (Alam et al., 2010). Despite the identification of these transcription factors, the molecular mechanism underlying bile salts sensing and T3SS2 signal propagation by V. parahaemolyticus and V. cholerae is unknown.

Here, we identify VtrC (VPA1333) from V. parahaemolyticus as a previously unrecognized component necessary for bile salt sensing and T3SS2 activation. vtrC is constitutively co-transcribed with vtrA as an overlapping 3’ open reading frame. Furthermore, we demonstrate that the periplasmic domains of the transmembrane proteins, VtrA and VtrC, form a functional complex that binds bile salts to activate VtrA’s cytoplasmic DNA binding domain, which in turn induces T3SS2 via the downstream transcription factor VtrB. The structure of VtrA/VtrC periplasmic domains reveals an obligate heterodimer where VtrC recruits structural elements from VtrA to complete a β-barrel with a hydrophobic inner chamber that binds bile salts. A co-crystal structure of the VtrA/VtrC heterodimer with a bile salt reveals that ligand binding occurs in the hydrophobic inner chamber of VtrC, similar to that found for the family of monomeric calycins. Mutations of residues within the hydrophobic chamber that are important for bile salt-binding disrupt bile salt activation of T3SS2. Collectively, we uncover a mechanism by which bacteria can sense bile salts, and reveal how an evolutionarily conserved receptor senses an environmental cue to induce the production of virulence factors.

Bile sensing plays a significant role in human infection caused by various enteric bacterial pathogens (Begley et al., 2005). Deciphering how bacteria use bile as a signal to regulate the expression of virulence genes is essential to our understanding of disease mechanisms and also can offer insights for designing novel therapeutic strategies. Bile salt-induced T3SSs are the primary virulence factor in many Vibrio species (Broberg et al., 2011). In this study, we uncovered a previously unidentified component of a widespread, conserved signaling platform that is essential for bile salt sensing and activation of virulence factors in Vibrios. Our biochemical data and the structural analysis demonstrated that the periplasmic domains of the inner membrane proteins VtrA and VtrC form a stable complex that functions as a receptor for bile salts.

The crystal structure of the VtrA/VtrC complex reveals that their periplasmic domains form a obligate heterodimer where VtrC folds into an open eight-stranded β-barrel and VtrA adopts a mixed α/β structure whereby one of its a β-strands contributes to the formation of the β-barrel. The VtrA/VtrC heterodimer fold is similar to members of the calycin superfamily, which contain eight to ten-stranded β-barrels that often bind hydrophobic molecules (Flower et al., 2000). Calycins have low sequence similarity, but relatively high structural conservation. They can be subdivided into several families based on structural details characteristic of each group (Figure 7) (Flower et al., 2000). Calycin families are markedly different in their distribution among biological kingdoms, cellular localization, and function, e.g.: (1) FABPs are cytosolic proteins involved in lipid homeostasis in animals, (2) MPIs are bacterial proteins that are secreted into the periplasm to inhibit metalloproteases, (3) lipocalins are found in both eukaryotes and prokaryotes, and they are mostly extracellular or attached to the outer membrane and bind a variety of ligands (Flower et al., 2000). In terms of structure, VtrC does not seem to cluster with any particular family and it forms its own clade (Figure 7). Although calycins have been found to form complexes with other proteins, our inspection of the available structures of complexes did not reveal any arrangement that resembled that of the VtrA/VtrC obligate heterodimer. This complex is unique for this family because it is formed from two proteins and it is a complex that transmits a signal upon binding its ligand.

Interestingly, members of the FABP family have been found to bind bile salts. This is exemplified by human and porcine ileal lipid binding proteins (ILBPs), and their NMR structures in complex with taurocholate (Kurz et al., 2003) and glycocholate (Lücke et al., 2000), respectively. Analogous to VtrC's disordered loop and short α-helix, FABPs have a helix-turn-helix motif that forms a lid over one side of the barrel and is proposed to act as a portal for ligand access (Storch and McDermott, 2009). In the case of FABPs, it has been suggested that ligand binding induces subtle conformational changes that promote protein-membrane or protein-protein interactions (Storch and McDermott, 2009). We observe a similar scenario for the VtrA/VtrC obligate heterodimer bound to bile salts where, upon binding bile salt, it appears an unstructured loop is translocated from the inside of the hydrophobic chamber to the outside of the chamber, where it becomes structured. Future studies will focus on how a conformational change during bile salt binding may induce a switch in the VtrA/VtrC interaction that will result in activation of the VtrA cytoplasmic transcription activator domain (Figure 9).

The closest example with regard to complex formation and function is the GrlR/GrlA complex, a regulator of the T3SS-encoding locus of enterocyte effacement (LEE) in enterohaemorrhagic and enteropathogenic E. coli strains (EHEC and EPEC, respectively) (Padavannil et al., 2013). GrlR is a cytosolic lipocalin that represses the LEE repressor GrlA by binding to its helix-turn-helix (HTH) domain. Similar to vtrA and vtrC, grlR and grlA are transcribed from a bicistronic operon, although the order of the lipocalin and transcriptional regulator genes is reversed in the latter pair. Analytical ultracentrifugation and the structure of GrlR in complex with the GrlA HTH domain revealed a heterotrimeric complex where a GrlR dimer binds one GrlA HTH monomer (PDB ID: 4KT5) (Padavannil et al., 2013). It has been shown that the inner cavity of GrlR binds lipids (Jobichen et al., 2009). However, the role of lipid binding in this system is unknown. Although GrlR and VtrC have analogous roles regulating T3SS through their interaction with a transcription factor, they do so from different cellular compartments at different stoichiometries, and their binding partners (GrlA HTH domain and VtrA periplasmic domain, respectively) have different folds.

Several signaling features of VtrA/VtrC appear to parallel two other pairs of regulatory proteins found in V. cholerae, ToxR/ToxS and TcpP/TcpH, which control the expression of numerous virulence factors including CT and TCP. First, the genes encoding the proteins in each pair are arranged in bicistronic operons (Miller et al., 1989; Häse and Mekalanos, 1998). Second, VtrA/ToxR/TcpP and VtrC/ToxS/TcpH have the same cellular localization and membrane topology (Miller et al., 1989; Häse and Mekalanos, 1998; Miller et al., 1987). Third, VtrC, ToxS and TcpH affect the activity of VtrA, ToxR and TcpP, respectively. On this last point, we showed that VtrA and VtrC form a complex in vivo both before and after T3SS2 activation and in vitro through their periplasmic domains.

Our data supports a model in which VtrA and VtrC form a complex both in the absence and presence of bile salts. Upon binding bile salts to the hydrophobic chamber in the VtrA/VtrC complex, the cytoplasmic DNA binding domain of VtrA is activated and in turn induces VtrB to activate the T3SS2 (Figure 10). The specific changes caused by binding bile salts and their effects on the VtrA DNA-binding domain are yet to be determined. Although VtrA contains no cysteine residues in its periplasmic domain, it is still possible that bile salts increase VtrA activity by inducing its dimerization or oligomerization, similar to what has been proposed for the ToxR/ToxS and TcpP/TcpH systems (Ottemann and Mekalanos, 1996; Yang et al., 2013).

Figure 10

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The VtrA/VtrC complex is highly conserved in a group of diverse Vibrionaceae family species, as well as in Moritella marina. While the bile salt-induced T3SS in V. cholerae non-O1/O139 strains is necessary for bacterial colonization and the primary cause of disease during infection (Chaand et al., 2015), the role of T3SS in G. hollisae infection is yet to be characterized. For other bacteria encoding vtrA, vtrC and vtrB, such as Photobacterium marinum and V. caribbeanicus, only a few T3SS related genes or no T3SS related genes are observed in their genomes, respectively. The differences between T3SS gene representation in these bacteria demonstrate this system's diversity and imply that the T3SS may be evolving in response to different living environments. Based on surrounding genes, this bile salt receptor complex may have adapted in other species to bind other hydrophilic ligands. For example, in the ten strains that lack a vtrB homolog but possess a vtrA/vtrC pair, the gene downstream of the vtrA/vtrC operon encodes a putative sphingomyelin phosphodiesterase. This enzyme is a hydrolase involved in the metabolism of sphingolipids (Goñi and Alonso, 2002) and is a putative virulence factor (Oda et al., 2012). It is tempting to hypothesize that the homologues of VtrA/VtrC in these bacteria function as a sensor for sphingolipids and regulate the induction of the downstream gene that produces sphingomyelin phosphodiesterase.

In conclusion, using microbial genetics and biophysics, we have identified and characterized a bile salt sensor that is widely distributed and used to induce virulence by many Vibrios. Additionally, we have found that a family of monomeric lipid binding calycin domain proteins has expanded to include an obligate heterodimer that binds bile salts and can be used to transmit a signal. To our knowledge, our study provides the first biochemical and structural analysis of a prokaryotic receptor involved in mediating a response to bile salts, a significant environmental cue during infection.

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[Editors’ note: this article was originally rejected after discussions between the reviewers, but the authors were invited to resubmit after an appeal against the decision.]

Thank you for submitting your work entitled "Bile Salt Receptor Complex Activates a Pathogenic Type III Secretion System" for consideration by eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Richard Losick as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Alejandro Buschiazzo (Reviewer #2).

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

The reviewers and editor found the work intriguing. However, there was unanimity in the feeling that the manuscript did not provide an understanding of bile recognition in the investigated signal transduction pathway. Moreover, there were several technical and conceptual issues that are detailed in the comments presented below.

Reviewer #1:

Li P et al. report the discovery of a new protein (VtrC) as an essential element to regulate type three secretion system 2 in Vibrio. VtrC is shown to form a complex with VtrA, which is responsive to bile (a known inductor of T3SS2, ultimately modulating virulence). VtrA and VtrB had already been identified as essential T3SS2 regulators, but the mechanism of bile sensing and signal transduction remains unknown, and this is the main question the authors want to address.

After identifying VtrC, the authors demonstrate that vtrA and vtrC belong to a single operon and, elegantly, that deletion of vtrC specifically affects T3SS2 and bile sensitivity.

VtrC is further characterized as a bitopic membrane protein, which led to uncovering periplasmic interaction with VtrA in vivo (co-IP). Not affecting vtrA transcription, VtrC appears to be critical for VtrA protein stability.

1) Why didn't the authors raise an antibody against the whole periplasmic (or intracellular) domain of VtrA? (VtrA might be expressed in the absence of VtrC, but that last short segment is being degraded, or that epitope is conformational and sensitive to VtrC binding?)

2) The crystal structure of the VtrA/VtrC complex represents an important contribution, confirming the association between both proteins.

Some concerns about the crystallographic data (the authors' feedback is important):

• for a 2-wavelength MAD phasing, 0.25 seems a suspiciously low FOM figure (what can you say about the three Se sites that were not found with direct methods, on the final refined model?)

• Rfree in principle seems high (almost 30%) for a 2.7Å resolution structure. No electron density is shown. Were there any difficulties throughout refinement? Is electron density good throughout the asymmetric unit? (please include a supplementary figure with best and worst ED map regions)

• Stereochemical rmsd figures for bond lengths and angles are extremely low. Why? Were extra-tight restraints used during refinement? if yes, why?

Please briefly address these points in the Materials and methods.

3) Are there other, alternative, heterodimer architectures (or yet, tetrameric organizations etc) in the cubic packing? Please cite the next best inter-protomer buried interface surface, ruling out otherwise ambiguous oligomeric choices.

4) The structural data don't seem to contribute all that much into a mechanistic understanding. Particularly, the ligand-binding site cannot be pinpointed as the authors admit. Did the authors attempt crystallizing the complex in the presence of TDC or other potential ligands? Make this explicit. Structurally assessing how VtrA-VtrC recognizes bile would be a major mechanistic insight (see next).

5) TDC is used as the sole ligand assayed by ITC, based on a previous report, but bile is really heterogeneous. The authors would benefit of incubating the complex with bile, then purifying and crystallizing (also MS can be helpful), attempting to see and/or identify ligands, which may not be TDC. Actually, the authors claim that TDC binds with nM affinity, which is somewhat misleading. KD is actually on the low microM range (i.e. nearer microM than nM, in contrast to most FABPs used as reference in Richieri et al. 2000 Biochemistry). "Nanomolar" should thus be changed to "low μM" in the Abstract and Discussion. Maybe other bile components bind tighter and are relevant physiologically?

6) Structural data would also be valuable to understand the critical role of interacting residues in the VtrA/VtrC interface. Structure-based mutants of vtrC can be analyzed on the vtrC-knockout background by the kind of functional assays that the authors have performed.

7) The major concern overall for the paper is indeed the mechanistic insight, which was the main question the authors sought to answer in the first place. Figure 9 is too vague: which bile component(s) binds physiologically? If it were TDC, where does it bind on the VtrA/VtrC complex? What are the characteristics of the signal-triggered rearrangement of VtrA (and/or VtrC)?

Alternative hypothesis: VtrC stabilizes (somehow) VtrA, but it is VtrA on its own that binds bile components and transduces the signal. Given that recombinant VtrA's periplasmic domain is available, it would be important to assess its own capacity to bind TDC (using ITC). And, if really no binding is detectable, did the authors attempt crystallizing the VtrA periplasmic domain on its own? This structure could well inform about VtrC-triggered rearrangements.

Reviewer #2:

This manuscript by Li et al. describes research that increases understanding of how bile induces Type Three Secretion in Vibrio parahaemolyticus. It had previously been shown that the proteins VtrA and VtrB were necessary for bile-mediated induction of the T3SS2 system, and VtrA activated VtrB in response to bile. This paper identifies a new component of this system, VtrC, that is in the membrane associated with VtrA. The authors crystalize the periplasmic interacting domains of VtrA and VtrC, in the absence of bile, and show the complex binds bile. This is a nice manuscript that definitely increases our understanding of this process, but I feel the authors overstep a bit in terms of the novelty of this work and their conclusions that they provide mechanistic insight into this process. Therefore, my enthusiasm is somewhat tempered.

1) The authors state that "the mechanism of bile sensing by these bacteria remains elusive". From the introduction, it would seem that not bile responsive proteins have been identified, or the impact of bile on these proteins elucidated. However, in Vibrio cholerae ToxT binding to bile inhibits its ability to bind target promoters, and as the authors state in the discussion bile has been shown to induce disulfide bonds in ToxR.

2) The authors don't really uncover the mechanism of VtrAC bile sensing, but just another important component of the system. For instance, I was surprised that the authors did not use the crystal structure to engineer mutations that would be predicted to disrupt the interaction or disrupt the binding to bile. Moreover, no structures are determined with bile, and they only identify the structure of the periplasmic domains, so how bile binding is transduced to changes in DNA binding affinity is not understood. They clearly make important advances, but this manuscript does not provide the mechanism of bile sensing.

[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]

Thank you for resubmitting your work entitled "Bile Salt Receptor Complex Activates a Pathogenic Type III Secretion System" for further consideration at eLife. Your revised article has been favorably evaluated by Richard Losick (Senior editor), a Reviewing editor, and two reviewers.

The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below.

There some questions about the processing of the diffraction data, which should be addressed before the manuscript can be accepted for publication. These questions are as follows:

The authors should be aware that the F432 cubic space group is extremely rare in protein crystal structures (~200 entries belonging to it, among the >10⁵ structures in the PDB). So, although possible to encounter, as with any unusual discovery, one should be extra cautious to avoid mistakes.

For the next few comments, you might want to check whether your space group assignment was correct (or could there be a form of pseudo-symmetry?). Pseudo-symmetry often results in regions of the alleged ASU with weak electron density and greater difficulty in model building. This is why I had asked previously about how good the electron density is, not only in the good parts, but also in the bad ones (the authors misunderstood replying only with the obvious: that density is better in the core and worse in the flexible solvent-exposed regions). A mistake in the space group could end up being worrisome in giving parts of the model wrong (even on top of parts that are right). The new structure in complex with TDC, is in part reassuring (a confirmation with different packing), but some figures of its own, also raise questions.

Issues that have remained unanswered:

1) Phasing FOM too low: 0.25 is suspicious for a MAD experiment. Could be explained because of too little anomalous signal (e.g. only two methionines for approx. 200 residues – probably a quite tough phasing scenario). Again, my point was to think (and test!) whether there is a lower symmetry possibility to integrate the data in, and then see if shelxd works better. Straightforward tests that you can also perform to scrutinize this SG assignment: a) use ccp4 program zanuda to analyze your cubic data, it will systematically test for lower compatible space group choices (and also perform some initial refinement for each, to have comparative figures); b) reintegrate the cubic data in P1, and perform mol replacement now that you have a solution (which is maybe completely right, or at least partially right for sure), searching for as many hetero-dimers as you calculate according to the triclinic unit cell volume: if a solution is found, than you can analyze if the packing indeed conforms to a such highly symmetric space group as F432.

2) Maybe it is cubic, end of story.

3) But, if alternative choices do appear, not only shelxd might give better FOM, but actually maps may turn out better (in the bad parts, if there were); final refined average B factors closer to Wilson B (this is a definitely strange behavior reported in your table); better Rmerge statistics (also high, not only in the high resolution shell, but also in the low res – I know high multiplicity is here at play, so I do not want to say this is necessarily wrong: just look closely and critically at it, to avoid mistakes).

4) I would strongly advise against including hydrogen atoms in the refinements. Else, explain really well why you think it's OK to include them. This goes also for the orthorhombic form with TDC. The validation reports seem clear in that you have included them: to support a refinement strategy with independent H atoms you would need ultra-high resolution (i.e. better than 1.2Å) or a neutron light source for the diffraction experiment.

5) The nominal resolution for the new orthorhombic structure is probably not 2.1Å: very low completeness of the data (57% high res shell). Please reconsider this. Actually, please elaborate as to why completeness drops significantly comparing the processed data vs those used for refinement.

It is suggested to reduce/simplify subsection “The VtrA/VtrC complex is an obligate heterodimer”; it's enough there to indicate that only 2 Se-Met out of the 5 present in the protein complex, were ultimately seen to be ordered in the refined model, hence contributing proportionally little to the measured anomalous signal in the first place (likely explaining the low FOM figure at the phasing step, previous to density modification).

In this context it is rather misleading to state that the 2 Se-Met "were used for refinement", actually you are probably talking here about their use to solve the substructure of anomalous scatterers for subsequent protein phasing purposes.

Reviewer #1:

Li P et al. report the discovery of a new protein (VtrC) as an essential element to regulate type three secretion system 2 in Vibrio. VtrC is shown to form a complex with VtrA, which is responsive to bile (a known inductor of T3SS2, ultimately modulating virulence). VtrA and VtrB had already been identified as essential T3SS2 regulators, but the mechanism of bile sensing and signal transduction remains unknown, and this is the main question the authors want to address.

After identifying VtrC, the authors demonstrate that vtrA and vtrC belong to a single operon and, elegantly, that deletion of vtrC specifically affects T3SS2 and bile sensitivity.

VtrC is further characterized as a bitopic membrane protein, which led to uncovering periplasmic interaction with VtrAin vivo (co-IP). Not affecting vtrA transcription, VtrC appears to be critical for VtrA protein stability.

1) Why didn't the authors raise an antibody against the whole periplasmic (or intracellular) domain of VtrA? (VtrA might be expressed in the absence of VtrC, but that last short segment is being degraded, or that epitope is conformational and sensitive to VtrC binding?)

We made a peptide antibody to the most antigenic region of VtrA found on its C-terminus and used this reagent in many of our studies. We agree with the reviewer that the possibility exists that the C-terminal fragment of VtrA could be degraded and we would miss this with our antibody. To avoid confusion, we have rephrased the following sentence "Taken together, VtrC appears to function upstream of VtrB through a mechanism that may involve stabilizing VtrA." to "Thus, VtrC appears to function upstream of VtrB via a signaling cascade including VtrA. The observed disappearance of VtrA in the absence of VtrC could support a direct physical interaction between VtrA and VtrC." This hypothesis is then validated by our structural studies with the VtrA/VtrC complex.

2) The crystal structure of the VtrA/VtrC complex represents an important contribution, confirming the association between both proteins.

Some concerns about the crystallographic data (the authors' feedback is important):

• for a 2-wavelength MAD phasing, 0.25 seems a suspiciously low FOM figure (what can you say about the three Se sites that were not found with direct methods, on the final refined model?)

We have included the following explanation in the Results:

“The structure was solved by multiple-wavelength anomalous dispersion phasing using anomalous signals from selenomethionine and refined to a resolution of 2.70 Å using native data from an isomorphous crystal. Two of the five methionines, VtrA Se-Met 210 at the interface with VtrC and VtrC Se-Met 49, were used for refinement. Another two methionines showed no electron density and we presume they are highly disordered because they are N-terminal methionines. The final missing Se-Met is Se-Met 115 from VtrC, which is in the disordered loop that covers the β-barrel.”

The quoted Figure of Merit (FOM) of 0.25 was calculated at the end of phasing and thus is not inflated due to assumptions made during density modification. The FOM after density modification was 0.88; this is clearly stated in the Materials and methods section, “Phase determination and structure refinement.

• Rfree in principle seems high (almost 30%) for a 2.7Å resolution structure. No electron density is shown. Were there any difficulties throughout refinement? Is electron density good throughout the asymmetric unit? (please include a supplementary figure with best and worst ED map regions)

Extensive efforts were made to appropriately model the native apo structure in the F432 space group. Several factors contribute to the R_free value observed in this structure, including the high solvent content (66%), the presence of a significant percentage of disordered and therefore unmodeled residues at the N-termini of both VtrA and VtrC as well as the disordered loop of VtrC (9% of all residues in the asymmetric unit), a lack of sidechain density for a significant number of residues on the solvent-exposed surface of the heterodimer, as well as significant differences in the mean B-values between VtrA and VtrC (62 Å2 vs. 77 Å2, respectively). Despite these issues, the observed R_free is within acceptable limits for the observed high resolution limit of the data (Kleywegt G.J. and Brünger A.T. (1996) Checking your imagination: applications of the free R value. Structure4:897-904). The “best” electron density map regions correspond to the hydrophobic core of each monomer, while the “worst” regions correspond to the surface of the heterodimer that is exposed to the bulk solvent.

• Stereochemical rmsd figures for bond lengths and angles are extremely low. Why? Were extra-tight restraints used during refinement? if yes, why?

Please briefly address these points in the Materials and methods.

Standard methods were used to refine the model in PHENIX, including the automated optimization of the weights for the X-ray observations/stereochemistry and X-ray observations/ADP (atomic displacement parameters). This optimization balances the results of minimization of R_work and R_free with the stereochemical and ADP restraints, and prevents chemically unreasonable geometry distortions. Given the medium-low resolution of the dataset plus the high solvent content (as discussed in the previous point) as well as the excellent geometry of the final model, it appears that PHENIX has done an appropriate job in this case.

3) Are there other, alternative, heterodimer architectures (or yet, tetrameric organizations etc) in the cubic packing? Please cite the next best inter-protomer buried interface surface, ruling out otherwise ambiguous oligomeric choices.

An analysis of the macromolecular interfaces found in the native apo structure in the F432 space group by the web server PDBePISA (http://www.ebi.ac.uk/pdbe/pisa/pistart.html) (E. Krissinel and K. Henrick (2007). 'Inference of macromolecular assemblies from crystalline state.'. J. Mol. Biol. 372, 774--797) was performed. Results indicate that the VtrA/VtrC heterodimer is the most stable quaternary structure in solution. The analysis yields a buried surface area of 2,300 Å2 between VtrA and VtrC, with ∆G^int = -9.6 kcal/mol (∆G^int is the free energy gain due to bringing the subunits together into a complex), ∆G^diss = 3.1 kcal/mol (∆G^diss is the free energy of dissociation), plus two salt bridges and ten hydrogen bonds at the interface. All other potential oligomeric assemblies, despite having similar or larger buried surface areas, had unfavorable (negative) values of ∆G^diss and were biologically incompatible with the requirement that all VtrA and VtrC proteins must be anchored into the same membrane by N-terminal helical segments on each protein. In addition, these alternative assemblies are not observed in the native bile-salt bound crystalline lattice, space group P2₁2₁2₁.

4) The structural data don't seem to contribute all that much into a mechanistic understanding. Particularly, the ligand-binding site cannot be pinpointed as the authors admit. Did the authors attempt crystallizing the complex in the presence of TDC or other potential ligands? Make this explicit. Structurally assessing how VtrA-VtrC recognizes bile would be a major mechanistic insight (see next).

We have solved the structure of the VtrA.VtrC complex bound to TDC and this has indeed provided “major mechanistic insight”.

5) TDC is used as the sole ligand assayed by ITC, based on a previous report, but bile is really heterogeneous. The authors would benefit of incubating the complex with bile, then purifying and crystallizing (also MS can be helpful), attempting to see and/or identify ligands, which may not be TDC.

We have included a new supplemental figure, Figure 5—figure supplement 1, showing the activation of T3SS2 by specific bile salts. Previously, Gotoh et al. (2010) established that bile salts are the component of bile that activates the T3SS2. We have recapitulated this data, and shown that the bile salt TDC is an activator of this system. Therefore, we used TDC for our biophysical analysis of the VtrA/VtrC complex.

Actually, the authors claim that TDC binds with nM affinity, which is somewhat misleading. KD is actually on the low microM range (i.e. nearer microM than nM, in contrast to most FABPs used as reference in Richieri et al. 2000 Biochemistry). "Nanomolar" should thus be changed to "low μM" in the Abstract and Discussion. Maybe other bile components bind tighter and are relevant physiologically?

In Richieri's paper, they tested FABPS from different sources against a variety of fatty acids by the ADIFAB fluorescence method. Depending on the specific FABP and fatty acid combination, Kd values varied from 2-367 nM. The affinity of Vtra/VtrC for TDC is comparable with the higher end of this range. Previous work by Gotoh at al. and new supplemental Figure 5—figure supplement 1 demonstrate that TDC is a physiologically relevant molecule for this system.

6) Structural data would also be valuable to understand the critical role of interacting residues in the VtrA/VtrC interface. Structure-based mutants of vtrC can be analyzed on the vtrC-knockout background by the kind of functional assays that the authors have performed.

We have addressed this comment with experiments in Figure 9 that show changing amino acids within the ligand-binding pocket disrupts bile salt mediated signaling, as this is the focus of this paper. Future studies will include mutations that address signal transduction mediated by bile salt binding; however, these studies are clearly beyond the scope of this paper.

7) The major concern overall for the paper is indeed the mechanistic insight, which was the main question the authors sought to answer in the first place. Figure 9 is too vague: which bile component(s) binds physiologically? If it were TDC, where does it bind on the VtrA/VtrC complex? What are the characteristics of the signal-triggered rearrangement of VtrA (and/or VtrC)?

Alternative hypothesis: VtrC stabilizes (somehow) VtrA, but it is VtrA on its own that binds bile components and transduces the signal. Given that recombinant VtrA's periplasmic domain is available, it would be important to assess its own capacity to bind TDC (using ITC). And, if really no binding is detectable, did the authors attempt crystallizing the VtrA periplasmic domain on its own? This structure could well inform about VtrC-triggered rearrangements.

We have solved the co-crystal structure of VtrA/VtrC with bile salts. As predicted, based on calycin domains, the bile salt binds in the hydrophobic chamber of the VtrA/VtrC complex. Rearrangements in the structure when binding TDC are observed and discussed. Mutant experiments with residues in the hydrophobic chamber corroborate the finding that bile salts bind in the hydrophobic pocket

Parenthetically, early in our studies we tried to see if recombinantly purified VtrA could bind bile salts using ITC. We saw no binding (Figure 1). After solving the structures of the VtrA/VtrC complex +/- bile salts and realizing it exists as an obligate heterodimer, we saw no reason to include this negative data.

Reviewer #3:

1) The authors state that "the mechanism of bile sensing by these bacteria remains elusive". From the introduction, it would seem that not bile responsive proteins have been identified, or the impact of bile on these proteins elucidated. However, in Vibrio cholerae ToxT binding to bile inhibits its ability to bind target promoters, and as the authors state in the discussion bile has been shown to induce disulfide bonds in ToxR.

We have excluded the general statement “bile sensing by these bacteria remains elusive”. We have included studies that have implicated bile salts in ToxT signaling. However, receptors involved in sensing bile salts and activating T3SS2 were unknown before this study.

2) The authors don't really uncover the mechanism of VtrAC bile sensing, but just another important component of the system. For instance, I was surprised that the authors did not use the crystal structure to engineer mutations that would be predicted to disrupt the interaction or disrupt the binding to bile. Moreover, no structures are determined with bile, and they only identify the structure of the periplasmic domains, so how bile binding is transduced to changes in DNA binding affinity is not understood. They clearly make important advances, but this manuscript does not provide the mechanism of bile sensing.

We have solved the co-crystal structure of VtrA/VtrC with bile salts. As predicted, it binds in the hydrophobic chamber of the VtrA/VtrC complex. Mutant experiments that corroborate this observation are also included in Figure 9.

Showing how bile binding is transduced to changes in DNA binding affinity of a transcription factor is work that we will address in the future and is out of the scope of this manuscript.

[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]

There some questions about the processing of the diffraction data, which should be addressed before the manuscript can be accepted for publication. These questions are as follows:

The authors should be aware that the F432 cubic space group is extremely rare in protein crystal structures (~200 entries belonging to it, among the >10⁵ structures in the PDB). So, although possible to encounter, as with any unusual discovery, one should be extra cautious to avoid mistakes.

For the next few comments, you might want to check whether your space group assignment was correct (or could there be a form of pseudo-symmetry?). Pseudo-symmetry often results in regions of the alleged ASU with weak electron density and greater difficulty in model building. This is why I had asked previously about how good the electron density is, not only in the good parts, but also in the bad ones (the authors misunderstood replying only with the obvious: that density is better in the core and worse in the flexible solvent-exposed regions).

The authors have now provided two figures, Author response image 1 and 2 illustrating good versus poor electron density in the structure, as exemplified by the lack of density for the high percentage of solvent-exposed side chains (solvent content of the lattice is 68%).

Author response image 1

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Author response image 2

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A mistake in the space group could end up being worrisome in giving parts of the model wrong (even on top of parts that are right). The new structure in complex with TDC, is in part reassuring (a confirmation with different packing), but some figures of its own, also raise questions.

The authors are acutely aware that F432 is a rather unusual space group, and spent much time and effort to rule out other possibilities. There is no twinning possible in the space group F432, but it is possible for lower symmetry space groups. A summary of the extensive analysis is now presented.

Scaling of data was performed also in the space group F23, a subgroup of F432, to test for the presence of twinning. If the space group is really F23, it could be merohedrally twinned and resemble F432. However, multiple tests indicate that in fact the data as scaled in F23 is NOT twinned tests (for more details on the nature of these tests, please see Padilla JE, Yeates TO. (2003) A statistic for local intensity differences: robustness to anisotropy and pseudo-centering and utility for detecting twinning. Acta Crystallogr D Biol Crystallogr 59:1124-30; Yeates TO. (1997) Detecting and overcoming crystal twinning. Methods Enzymol 276:344-58; Yeates TO (1988). Simple statistics for intensity data from twinned specimens. Acta Crystallogr A44:142-4.) Here are the statistics as reported in the Xtriage routine of PHENIX:

a) <I²>/<I>² = 2.113 (untwinned: 2.0, perfect twin: 1.5)

b) <F>²/<F²> = 0.771 (untwinned: 0.785, perfect twin 0.885)

c) <|E²-1|> = 0.755 (untwinned: 0.736, perfect twin 0.541)

d) <|L|> = 0.505 (untwinned: 0.500, perfect twin 0.375)

e) <L²> = 0.343 (untwinned: 0.333, perfect twin 0.200)

f) In addition, the output from PHENIX indicated that the point group of the lattice is F432, and explicitly stated there was no evidence that the lattice displays pseudosymmetry.

Pseudosymmetry typically manifests as systematically weak classes of reflections (for example, h + k = 2n reflections that are systematically weak) (Zwart P.H., Grosse-Kunstleve R. W., Lebedev A. A., Murshudov G. N., Adams P. D. (2008) Surprises and pitfalls arising from (pseudo)symmetry. Acta Crystallogr D Biol Crystallogr 64:99-107; Li W., Ma C., Guan R., Xu Y., Tomchick D.R., Rizo J. (2011) The crystal structure of a Munc13 C-terminal module exhibits a remarkable similarity to vesicle tethering factors. Structure, 19:1443-1455). When the data is scaled and merged in HKL3000 in Laue class m3 (i.e., space group F23), there is no sign of pseudosymmetry in the reflections (i.e., no systematically weak classes of reflections are observed), and the discrepancy indices (i.e., the “R-factors”) are virtually indistinguishable from those obtained when the data is scaled and merged in Laue class m3m (i.e., space group F432). Thus we conclude that the correct space group is in fact F432.

Issues that have remained unanswered:

1) Phasing FOM too low: 0.25 is suspicious for a MAD experiment. Could be explained because of too little anomalous signal (e.g. only two methionines for approx. 200 residues – probably a quite tough phasing scenario). Again, my point was to think (and test!) whether there is a lower symmetry possibility to integrate the data in, and then see if shelxd works better.

The authors have extensive experience in SAD/MAD phasing of SeMet-substituted soluble proteins, especially with projects that have low anomalous signal and significant radiation decay of the signal. This particular phasing experiment utilized 1 Se site for 108 amino acids (as density for only 216 amino acids was observed and modeled by the final rounds of refinement), hence the chosen phasing method was MAD and not SAD. Data processing and phasing was performed in the graphical user interface of HKL3000, with autocorrections implemented to correct for systematic errors such as radiation damage and absorption, and to apply B-factor sharpening (Borek D, Dauter Z, Otwinowski Z. (2013) Identification of patterns in diffraction intensities affected by radiation exposure. J Synchrotron Radiat 20:37-48.). We have found the multiple corrections applied in the autocorrections routine of HKL3000 to be critical to our success in phasing numerous difficult anomalous phasing projects. In this case, only the first wavelength (anomalous peak) was chosen for the heavy atom search, as more extensive radiation damage had occurred during the data collection for the second wavelength (inflection point). We performed a search for heavy atom positions to a resolution of 4.0 Å with SHELXD which identified two Se positions with correlation coefficients: CC_All = 52.9% and CC_Weak = 31.2%. It is difficult for the authors to understand what else they could have done with their data that could have resulted in SHELXD being able to locate additional selenium sites. Density for only two methionines was observed in the map from the final rounds of model refinement for the native protein (due to disorder of the N-terminal methionines from both monomers of the heterodimer, and the remaining missing methionine is located in the short disordered loop of five residues in VtrC). Both wavelengths were used for phasing in MLPHARE, with a resulting FOM of 0.25 for the complete resolution range from 35.7 – 2.60 Å. This compares well with the estimate of anomalous signal calculated in PHENIX:

Author response image 3

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In this table, the FOM estimate if the sub-structure (i.e., the Se sites) is solved is 0.3 (+/- 0.1). The P(Substr) is the estimate of probability that the sub-structure can be found with the heavy atom routine HYSS using the anomalous peak data (Terwilliger TC, Bunkóczi G, Hung L-W, Zwart PH, Smith JL, Akey DL, Adams PD (20160 Can I solve my structure by SAD phasing? Anomalous signal in SAD phasing. Acta Crystallogr D Biol Crystallogr 72:346-358).

The FOM after density modification in the program Parrot was 0.88, which was expected given that the solvent content of the lattice is 68%. The resulting map was sufficient for an automatic model building routine (Buccaneer) to accurately trace and assign sequence to 200 of 238 possible amino acids, or to 93% of all residues in the final model. Thus we believe that this model and space group are correct.

Straightforward tests that you can also perform to scrutinize this SG assignment: a) use ccp4 program zanuda to analyze your cubic data, it will systematically test for lower compatible space group choices (and also perform some initial refinement for each, to have comparative figures); b) reintegrate the cubic data in P1, and perform mol replacement now that you have a solution (which is maybe completely right, or at least partially right for sure), searching for as many hetero-dimers as you calculate according to the triclinic unit cell volume: if a solution is found, than you can analyze if the packing indeed conforms to a such highly symmetric space group as F432.

The authors performed this test, and a screenshot of the log file is shown below. The space group F432 appears to be the correct one (Zanuda reports larger R-free values than reported for the final round of model refinement, as it makes no TLS B-factor corrections to the model).

Author response image 4

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2) Maybe it is cubic, end of story.

This suggestion is redundant to the analysis performed in Zanuda and thus the authors have not performed this test.

3) But, if alternative choices do appear, not only shelxd might give better FOM, but actually maps may turn out better (in the bad parts, if there were); final refined average B factors closer to Wilson B (this is a definitely strange behavior reported in your table); better Rmerge statistics (also high, not only in the high resolution shell, but also in the low res – I know high multiplicity is here at play, so I do not want to say this is necessarily wrong: just look closely and critically at it, to avoid mistakes).

An extensive discussion with one of the authors of HKL3000 (Zbyszek Otwinowski) reveals the nature of the confusion regarding the Wilson B-factor. The data was scaled in HKL3000 using the autocorrections routine, which performs extensive B-factor sharpening; only the sharpened B-factors were originally reported. The unsharpened Wilson B-factors for all datasets are now included in Table 1. For the SeMet and native F432 structures, the unsharpened Wilson B-factors are 77.6 to 79.0, very similar with the observed mean B-values for the refined model.

It is a well-known consequence of high multiplicity (in the case of the native model, 36-fold) that the values for Rmerge will be artificially higher than expected, even though the merged data are improved by averaging more observations. This is the reason that reported in Table 1 are values for Rpim, the precision indicating R-factor (Evans P.R. (2011) An introduction to data reduction: space group determination, scaling and intensity statistics. Acta Crystallogr D Biol Crystallogr 67:282-92.), which estimates the data quality after merging. These values are roughly a factor of 6-fold lower than the Rmerge values, and we think these values more accurately reflect the quality of the native data set. In addition, the CC_1/2 value for the highest resolution shell (0.934) generously exceeds that recommended as a suitable high-resolution cutoff limit (Diederichs K. and Karplus P.A. (2012) Linking crystallographic model and data quality. Science 336:1030-1033).

4) I would strongly advise against including hydrogen atoms in the refinements. Else, explain really well why you think it's OK to include them. This goes also for the orthorhombic form with TDC. The validation reports seem clear in that you have included them: to support a refinement strategy with independent H atoms you would need ultra-high resolution (i.e. better than 1.2Å) or a neutron light source for the diffraction experiment.

Modern methods of macromolecular crystallographic refinement encourage the inclusion of hydrogen atoms at ALL resolutions. These hydrogen atoms are not located independently in an electron density map and refined independently of the non-hydrogen atoms in the model; rather, they are placed in geometrically idealized conformations with relation to the non-hydrogen atoms they are covalently bonded to and during positional refinement they are tied to their bonded atoms. These are thus known as “riding hydrogen atoms” and they have been used for decades in small-molecule refinements (especially in cases where hydrogen atoms are not explicitly observed in the electron density maps). Since 2010 the program authors of PHENIX have encouraged users to incorporate riding hydrogen atoms into their refinement protocols (Adams P.D., et al. (2010) PHENIX – a comprehensive Python-based system for macromolecular structure solution. Acta Crystallographica D Biol Crystallogr 66:213–221; Afonine PV, Mustyakimov M, Grosse-Kunstleve RW, Moriarty NW, Langan P, Adams PD (2010) Joint X-ray and neutron refinement with phenix.refine. Acta Crystallogr D Biol Crystallogr 66:1153-63.), and in fact, REFMAC adds the riding hydrogen atoms by default (http://www.ccp4.ac.uk/html/refmac5/keywords/restraints.html). The reasons for including riding hydrogen atoms in refinement are two-fold: 1) the hydrogen atoms actually exist in the molecules and contribute to the X-ray scattering at low resolution, so their inclusion in the refinement improves the agreement between F_obsand F_calc at low resolution; and 2) their inclusion improves the geometry of the refined model, as they are used to analyze clashes due to van der Waals repulsions. Jane and David Richardson have shown the utility of this method with the MolProbity server, which requires hydrogen atoms for the all-atom contact analysis it performs for validation of macromolecular structures (Chen et al. (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallographica D Biol Crystallogr 66:12-21.).

The two major refinement packages handle riding hydrogen atoms differently. REFMAC utilizes implicit hydrogen atoms and does not automatically write them out to the resulting PDB file (although the user may choose to explicitly to write them out), and PHENIX automatically includes them in the resulting PDB file. The user would actively need to remove the hydrogen atoms from a file generated by the PHENIX refinement program prior to PDB deposition. The authors of this work have chosen to include all atoms used to calculate the structure factors that are deposited along with the model coordinates in the PDB, thus statistics for the riding hydrogen atoms appear in the PDB validation report.

5) The nominal resolution for the new orthorhombic structure is probably not 2.1Å: very low completeness of the data (57% high res shell). Please reconsider this. Actually, please elaborate as to why completeness drops significantly comparing the processed data vs those used for refinement.

This dataset displays significant anisotropy, which results in the relatively low completeness in the highest resolution shell during data collection (79.4%). Anisotropic diffraction was corrected to adjust the error model and to compensate for a radiation-induced increase of non-isomorphism within the crystal. These corrections were applied to the data during scaling in the autocorrections routine of HKL3000 and thus are responsible for the difference in completeness between the data collection and refinement (Borek D, Dauter Z, Otwinowski Z. (2012) Identification of patterns in diffraction intensities affected by radiation exposure. J Synchrotron Radiat 20:37-48; Borek D, Cymborowski M, Machius M, Minor W, Otwinowski Z. (2010) Diffraction data analysis in the presence of radiation damage. Acta Crystallogr D Biol Crystallogr 66:426-36; Borek D, Ginell SL, Cymborowski M, Minor W, Otwinowski Z. (2007) The many faces of radiation-induced changes. J Synchrotron Radiat 14:24-33.).

The resolution chosen as the refinement limit (2.10 Å) was chosen carefully and is consistent with current practices.

a) The I/σ(I) is greater than 1.0 and less than 3.0;

b) The percentage completeness in the highest resolution shell is greater than 50%;

c) The CC_1/2 in the highest resolution shell is 0.754, and greater than 0.5 (this value for all datasets has been included in Table 1;

d) The multiplicity in the highest resolution shell is greater than 2.0;

e) The electron density map, the refinement statistics and the model stereochemical parameters degrade in quality when the highest resolution limit used for refinement is 2.15 Å.

It is current practice to include data from resolution shells that in the past were ignored due to a high percentage of weak data and/or lower than 100% completeness due to anisotropy (Diederichs K. and Karplus P.A. (2012) Linking crystallographic model and data quality. Science 336:1030-1033; Weiss M.S. (2001) Global indicators of X-ray data quality. J Appl Cryst 34:130-135; Diederichs K. and Karplus P.A. (1997) Improved R-factors for diffraction data analysis in macromolecular crystallography. Nat Struct Mol Biol 4:269-274.). In this case, inclusion of the data in resolution shells to 2.10 Å actually improves the model and refinement statistics, which are excellent, and thus the authors have not changed the upper resolution limit for the structure with the bile salt.

It is suggested to reduce/simplify subsection “The VtrA/VtrC complex is an obligate heterodimer”; it's enough there to indicate that only 2 Se-Met out of the 5 present in the protein complex, were ultimately seen to be ordered in the refined model, hence contributing proportionally little to the measured anomalous signal in the first place (likely explaining the low FOM figure at the phasing step, previous to density modification).

In this context it is rather misleading to state that the 2 Se-Met "were used for refinement", actually you are probably talking here about their use to solve the substructure of anomalous scatterers for subsequent protein phasing purposes.

Indeed the wording that should have been used in this case was “Two of the five methionines, VtrA Se-Met 210 at the interface with VtrC and VtrC Se-Met 49, were used for phasing.” This sentence, plus the following two sentences, were added in response to the previous reviewer questions regarding how the structure could have been phased with only two selenomethionine sites. Thus we will replace these three sentences with the following shorter, clearer sentences:

“Two of the five selenomethionines were used for phasing. The two N-terminal methionines and Se-Met 115 in the loop that covers the β barrel of VtrC were not observed in the final electron density map and were not included in the final model.”

Author details

1. Peng Li

   Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   PL, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Giomar Rivera-Cancel

   Competing interests

   The authors declare that no competing interests exist.

2. Giomar Rivera-Cancel

   Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   GR-C, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Peng Li

   Competing interests

   The authors declare that no competing interests exist.

3. Lisa N Kinch

   Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   LNK, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

4. Dor Salomon

   Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   DS, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

5. Diana R Tomchick

   1. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, United States
   2. Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   DRT, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-7529-4643

6. Nick V Grishin

   1. Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, United States
   2. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, United States
   3. Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   NVG, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

7. Kim Orth

   1. Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, United States
   2. Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, United States
   3. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   KO, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   kim.orth@utsouthwestern.edu

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-0678-7620

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