Structural predictions have reached unprecedented accuracy. They leverage sequence-specific data to capture all potential interactions a sequence has evolved to fulfill. AlphaFold derives information from three sources: learned parameters capturing intrinsic amino acid secondary structure and environment propensity; models of related proteins providing structural templates; and aligned sequences encoding profiles and concerted evolutionary changes of residues involved in contacts. However, function demands dynamic changes; hence not all possible interactions can coexist simultaneously. Comprehensive information entails contradictions, which resolved in favor of the better-informed structure will silence less stable states and associations. Here, we introduce a method using all three channels to include prior knowledge: site-specific variants, predefined alignments and templates. Selecting information relevant to a particular state delimits the functional context of a prediction. Our program VAIRO allows us to rescue asymmetric and weaker interactions to complete the view of molecular assemblies in the architecture of a bacterial surface layer, and reveals otherwise inaccessible dynamic states in a pneumococcal multimeric membrane protein complex. VAIRO is distributed via the python package index (PyPI) (https://pypi.org/project/vairo) and the code is also available on Github (https://github.com/arcimboldo-team/vairo).

Crystallography provides structural evidence of macromolecules in atomic detail. However, the atomic structure is not the direct outcome of the experiment. Diffraction data need to be processed and the phase problem must be solved to visualize the map from which an atomic model of the macromolecule is interpreted and iteratively improved. Despite this complex process from sample to scientific answer, crystallography is widely accessible in biochemical and biological research. Easy access to the experimental set-ups, free software for academic use and complimentary analytical computing, supported by automation and expert assistance, makes crystallography available to non-crystallographers. This Primer offers a practical and rational introduction to macromolecular crystallography, whether to engage directly or to critically assess results, with a focus on understanding the diffraction data, solving the phase problem, building and refining the atomic model, and interpreting the resulting atomic structure. We provide an overview of what crystallography can achieve, the key decisions and trade-offs involved, and how to evaluate outcomes effectively. This Primer offers a practical and rational introduction to macromolecular crystallography, whether to engage directly with or to critically assess results, with a focus on understanding the diffraction data, solving the phase problem, building and refining the atomic model, and interpreting the resulting atomic structure.

S-layers are crystalline arrays found on bacterial and archaeal cells. Lactobacillus is a diverse family of bacteria known especially for potential gut health benefits. This study focuses on the S-layer proteins from Lactobacillus acidophilus and Lactobacillus amylovorus common in the mammalian gut. Atomic resolution structures of Lactobacillus S-layer proteins SlpA and SlpX exhibit domain swapping, and the obtained assembly model of the main S-layer protein SlpA aligns well with prior electron microscopy and mutagenesis data. The S-layer’s pore size suggests a protective role, with charged areas aiding adhesion. A highly similar domain organization and interaction network are observed across the Lactobacillus genus. Interaction studies revealed conserved binding areas specific for attachment to teichoic acids. The structure of the SlpA S-layer and the suggested incorporation of SlpX as well as its interaction with teichoic acids lay the foundation for deciphering its role in immune responses and for developing effective treatments for a variety of infectious and bacteria-mediated inflammation processes, opening opportunities for targeted engineering of the S-layer or lactobacilli bacteria in general.

Vacuolar sorting receptors (VSRs) are type I membrane proteins crucial for seed germination and plant development. While VSR trafficking has been extensively studied, the mechanism of cargo binding and release by the N-terminal lumenal region is less understood. We have elucidated the crystal structures of unloaded and cargo-loaded forms of the lumenal region of VSR1 containing the protease-associated domain, the Central domain and epidermal growth factor-like repeats. Calcium coordination induces remodeling of the linkers between domains, triggering large conformational changes that expose two binding sites, one across the PA and Central domain and a second site in the Central domain. Our findings provide a mechanistic model for cargo binding in a calcium rich environment, where VSR is locked in a conformation exposing the cargo binding sites, while cargo release is favoured by lower calcium concentrations and trimer formation. These results advance our current knowledge on VSRs and will inform future studies on vacuolar trafficking and cargo binding/release. ### Competing Interest Statement The authors have declared no competing interest.

Symmetry in nature often emerges from self-assembly processes and serves a wide range of functions. Cell surface layers (S-layers) form symmetrical lattices on many bacterial and archaeal cells, playing essential roles such as facilitating cell adhesion, evading the immune system, and protecting against environmental stress. However, the experimental structural characterization of these S-layers is challenging due to their self-assembly properties and high sequence variability. In this study, we introduce the SymProFold pipeline, which utilizes the high accuracy of AlphaFold-Multimer predictions to derive symmetrical assemblies from protein sequences, specifically focusing on two-dimensional S-layer arrays and spherical viral capsids. The pipeline tests all known symmetry operations observed in these systems (p1, p2, p3, p4, and p6) and identifies the most likely symmetry for the assembly. The predicted models were validated using available experimental data at the cellular level, and additional crystal structures were obtained to confirm the symmetry and interfaces of several SymProFold assemblies. Overall, the SymProFold pipeline enables the determination of symmetric protein assemblies linked to critical functions, thereby opening possibilities for exploring functionalities and designing targeted applications in diverse fields such as nanotechnology, biotechnology, medicine, and materials and environmental sciences.

PaaX is a transcriptional repressor of the phenylacetic acid (PAA) catabolic pathway, a central route for bacterial aerobic degradation of aromatic compounds. Induction of the route is achieved through the release of PaaX from its promoter sequences by the first compound of the pathway, phenylacetyl-coenzyme A (PA-CoA). We report the crystal structure of PaaX from Escherichia coli W. PaaX displays a novel type of fold for transcription regulators, showing a dimeric conformation where the monomers present a three-domain structure: an N-terminal winged helix-turn-helix domain, a dimerization domain similar to the Cas2 protein and a C-terminal domain without structural homologs. The domains are separated by a crevice amenable to harbour a PA-CoA molecule. The biophysical characterization of the protein in solution confirmed several hints predicted from the structure, i.e. its dimeric conformation, a modest importance of cysteines and a high dependence of solubility and thermostability on ionic strength. At a moderately acidic pH, the protein formed a stable folding intermediate with remaining α-helical structure, a disrupted tertiary structure and exposed hydrophobic patches. Our results provide valuable information to understand the stability and mechanism of PaaX and pave the way for further analysis of other regulators with similar structural configurations.

Density modification is a standard step to provide a route for routine structure solution by any experimental phasing method, with single-wavelength or multi-wavelength anomalous diffraction being the most popular methods, as well as to extend fragments or incomplete models into a full solution. The effect of density modification on the starting maps from either source is illustrated in the case of SHELXE . The different modes in which the program can run are reviewed; these include less well known uses such as reading external phase values and weights or phase distributions encoded in Hendrickson–Lattman coefficients. Typically in SHELXE , initial phases are calculated from experimental data, from a partial model or map, or from a combination of both sources. The initial phase set is improved and extended by density modification and, if the resolution of the data and the type of structure permits, polyalanine tracing. As a feature to systematically eliminate model bias from phases derived from predicted models, the trace can be set to exclude the area occupied by the starting model. The trace now includes an extension into the gamma position or hydrophobic and aromatic side chains if a sequence is provided, which is performed in every tracing cycle. Once a correlation coefficient of over 30% between the structure factors calculated from such a trace and the native data indicates that the structure has been solved, the sequence is docked in all model-building cycles and side chains are fitted if the map supports it. The extensions to the tracing algorithm brought in to provide a complete model are discussed. The improvement in phasing performance is assessed using a set of tests.

Crystallography at low resolution must determine the atomic model from less experimental observations, which is challenging in the absence of a model. In addition, model bias is more severe when independent experimental data are scarce. Our methods solve the phase problem by combining the location of accurate model fragments using Phaser with density modification and interpretation of the resulting maps using SHELXE. From a partial, correct structure, the density modification process and the stereochemical constraints draw the rest of the structure, validating the result. This same principle is now exploited at low resolution. Coiled coils are important, ubiquitous structures but notoriously difficult to phase and to predict. Both correct solutions and incorrect ones are poorly discriminated by the crystallographic figures of merit as long as helices are correctly oriented. We incorporate coiled-coil verification, designed to set up competing, incompatible structural hypotheses to probe both the results and establish the power of the data to discriminate them. Efficiency of coiled-coil phasing and validation in test cases from 3 to 4 Å is demonstrated in ARCIMBOLDO_LITE, placing single helices, and in ARCIMBOLDO_SHREDDER, with fragments derived from AlphaFold models. SHELXE tracing at low resolution has been enhanced, maintaining its local character but extending the environment assessment. For non-helical structures, verification is demonstrated in the fragment location process. Its use is exemplified with the solution of the VSR1 structure at 3.5 Å, depending on LLG optimization and the emergence of new features in the electron density. Relying on verification, we have extended the use of the ARCIMBOLDO software to low resolution.

ATP-binding cassette (ABC) transport systems are crucial for bacteria to ensure sufficient uptake of nutrients that are not produced de novo or improve the energy balance. The cell surface of the pathobiont Streptococcus pneumoniae (pneumococcus) is decorated with a substantial array of ABC transporters, critically influencing nasopharyngeal colonization and invasive infections. Given the auxotrophic nature of pneumococci for certain amino acids, the Ami ABC transporter system, orchestrating oligopeptide uptake, becomes indispensable in host compartments lacking amino acids. The system comprises five exposed Oligopeptide Binding Proteins (OBPs) and four proteins building the ABC transporter channel. Here, we present a structural analysis of all the OBPs in this system. Multiple crystallographic structures, capturing both open and closed conformations along with complexes involving chemically synthesized peptides, have been solved at high resolution providing insights into the molecular basis of their diverse peptide specificities. Mass spectrometry analysis of oligopeptides demonstrates the unexpected remarkable promiscuity of some of these proteins when expressed in Escherichia coli, displaying affinity for a wide range of peptides. Finally, a model is proposed for the complete Ami transport system in complex with its various OBPs. We further disclosed, through in silico modelling, some essential structural changes facilitating oligopeptide transport into the cellular cytoplasm. Thus, the structural analysis of the Ami system provides valuable insights into the mechanism and specificity of oligopeptide binding by the different OBPs, shedding light on the intricacies of the uptake mechanism and the in vivo implications for this human pathogen.

Full text Figures and data Side by side Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The seventh pandemic of the diarrheal cholera disease, which began in 1960, is caused by the Gram-negative bacterium Vibrio cholerae. Its environmental persistence provoking recurring sudden outbreaks is enabled by V. cholerae’s rapid adaption to changing environments involving sensory proteins like ToxR and ToxS. Located at the inner membrane, ToxR and ToxS react to environmental stimuli like bile acid, thereby inducing survival strategies for example bile resistance and virulence regulation. The presented crystal structure of the sensory domains of ToxR and ToxS in combination with multiple bile acid interaction studies, reveals that a bile binding pocket of ToxS is only properly folded upon binding to ToxR. Our data proposes an interdependent functionality between ToxR transcriptional activity and ToxS sensory function. These findings support the previously suggested link between ToxRS and VtrAC-like co-component systems. Besides VtrAC, ToxRS is now the only experimentally determined structure within this recently defined superfamily, further emphasizing its significance. In-depth analysis of the ToxRS complex reveals its remarkable conservation across various Vibrio species, underlining the significance of conserved residues in the ToxS barrel and the more diverse ToxR sensory domain. Unravelling the intricate mechanisms governing ToxRS’s environmental sensing capabilities, provides a promising tool for disruption of this vital interaction, ultimately inhibiting Vibrio’s survival and virulence. Our findings hold far-reaching implications for all Vibrio strains that rely on the ToxRS system as a shared sensory cornerstone for adapting to their surroundings. Editor's evaluation This study provides important insights into the structure and mechanism of the sensory protein complex ToxR/S that is associated with the survival and virulence of the cholera pathogen. The structural studies are solid and supported by a series of biophysical experiments revealing a split, periplasmic protein binding interface for bile acid. Results are of interest to protein biochemistry and pharmacology where they may open new routes for the treatment of cholera disease. https://doi.org/10.7554/eLife.88721.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Cholera is a contagious diarrheal disease that leads to about 20,000 to 140,000 yearly deaths. It is caused by a bacterium called Vibrio cholerae, which can survive in harsh conditions and many environments. It often contaminates water, where it lives in an energy-conserving mode. But when humans consume Vibrio cholerae-contaminated water or food, the bacterium can sense its new environment and switch into a high-energy consuming state, causing fever, diarrhea, and vomiting. Vibrio cholerae recognizes bile acid in the human stomach, which signals that the bacterium has reached ideal conditions for causing disease. So far, it has been unclear, how exactly the bacterium detects bile acid. Understanding how these bacteria sense bile acid, could help scientists develop new ways to prevent cholera outbreaks or treat infections. Gubensäk et al. analysed two proteins from the Vibrio cholerae bacterium, called ToxR and ToxS, which are located below the bacteria’s protective membrane. More detailed analyses showed that the two proteins bind together, forming a bile-binding pocket. When correctly assembled, this bile-sensing machine detects bile concentrations in the body, allowing the bacterium to adapt to the local conditions. Using crystal structures, a series of interaction studies, and modeling software, Gubensäk et al. detailed step-by-step how the two proteins sense bile acid and help the bacteria adapt and thrive in the human body. The results confirm the results of previous studies that implicated ToxR and ToxS in bile sensing and provide new details about the process. Scientists may use this information to develop new ways to interfere with the bacteria’s bile-sensing and gut adaptation processes. They may also use the information to screen for existing drugs that block bile sensing and then test as cholera treatments or prevention strategies in clinical trials. New cholera treatment or prevention approaches that don't rely on antibiotics may help public health officials respond to growing numbers of cholera outbreaks and to prevent the spread of antibiotic-resistant bacteria. Introduction The Gram-negative bacterium Vibrio cholerae is the causative agent of the diarrheal cholera disease, which is pandemic since 1960 (Hu et al., 2016). Its dangerousness is highlighted by its sudden outbreaks and environmental persistence causing 21 000–143,000 000 deaths worldwide per year (Ali et al., 2015) including long-lasting environmental and economic damage (Kanungo et al., 2022). V. cholerae exhibits a life cycle between dormant and virulent state, enabled by its rapid adaption to changing environments (Almagro-Moreno et al., 2015b; Bari et al., 2013). This survival mechanism is maintained via sensory proteins reacting to environmental conditions and substances (DiRita et al., 1991; Hung and Mekalanos, 2005). For entero-pathogens like V. cholerae, bile acid represents one of the major components for virulence activation, pressuring survival strategies (Hung et al., 2006; Hung and Mekalanos, 2005; Li et al., 2016; Midgett et al., 2017). ToxR is a transmembrane transcription factor (Figure 1—figure supplement 1) involved in the regulation of numerous genes, not only virulence associated, and can function as an activator, co-activator and repressor (Bina et al., 2003; Champion et al., 1997; Lee et al., 2000; Morgan et al., 2011; Skorupski and Taylor, 1997; Wang et al., 2002; Welch and Bartlett, 1998). ToxR periplasmic domain is proposed to act as environmental sensor, being able to bind bile acids (Midgett et al., 2017; Midgett et al., 2020) and consequently activate transcription with its cytoplasmic DNA binding domain (Gubensäk et al., 2021a; Morgan et al., 2011; Morgan et al., 2019; Pfau and Taylor, 1996; Withey and DiRita, 2006), thus inducing a switch of outer membrane proteins from OmpT to OmpU (Simonet et al., 2003; Wibbenmeyer et al., 2002). Since OmpU is more efficient in excluding bile salts due to its negatively charged pore (Duret and Delcour, 2006; Simonet et al., 2003), bile-induced ToxR activation enables V. cholerae survival in the human gut (Wibbenmeyer et al., 2002). ToxS is built of a periplasmic, a transmembrane and a short cytoplasmic region (Figure 1—figure supplement 1). The periplasmic domain of ToxS (ToxSp) interacts with the periplasmic domain of ToxR (ToxRp) forming a stable heterodimer (ToxRSp; Gubensäk et al., 2021b) thereby protecting ToxR from periplasmic proteolysis and enhancing its activity (Almagro-Moreno et al., 2015a; Almagro-Moreno et al., 2015c; Gubensäk et al., 2021b; Lembke et al., 2018; Pennetzdorfer et al., 2019). The exact mechanism of ToxR functionality is not clear yet, it was proposed that ToxR binds DNA as a homodimer at the so called ‘tox-boxes’ which represent direct repeat DNA motifs (Crawford et al., 1998; Goss et al., 2013; Krukonis and DiRita, 2003; Ottemann and Mekalanos, 1996; Pfau and Taylor, 1996; Withey and DiRita, 2006). Recently, it was shown that ToxR uses a topological DNA recognition mechanism by recognizing DNA structural elements rather than base sequences (Canals et al., 2023). Also, it is suggested that by multiple binding events of ToxR to promoter regions, membrane attached transcription regulation of ToxR is enabled (Canals et al., 2023). The versatile functionality of ToxR suggests a general role of ToxRS in Vibrio strains, for example the sensing and consequent adaption to changing environmental conditions (Chen et al., 2018; Provenzano et al., 2000). Bile significantly alters the virulence factor production. Nevertheless, the exact mechanism remains unclear. Studies showed opposite outcomes in regard of up- or downregulation of virulence factors by bile (Bina et al., 2021; Gupta and Chowdhury, 1997; Hung and Mekalanos, 2005; Midgett et al., 2017; Xue et al., 2016; Yang et al., 2013). On the one hand a bile induced reduction of virulence factor production was observed (Gupta and Chowdhury, 1997) via inhibiting ToxT DNA binding ability (Plecha et al., 2015). On the other hand bile seems to enhance TcpP and ToxR activity (Yang et al., 2013) and even induce ToxR ability to activate ctx without ToxT in classical biotype V. cholerae strains (Hung and Mekalanos, 2005). Nevertheless, direct activation of ctx by ToxRS by bile acids is dependent on ctx promotors, differing between strains (Hung and Mekalanos, 2005). Concluding, the effect of bile on V. cholerae virulence seems to be complex and probably dependent on multiple other factors for example calcium concentration and oxygen levels (Hay et al., 2017; Sengupta et al., 2014). The presented crystal structure of sensory domains of V. cholerae ToxR and ToxS reveals ToxS as a main environmental sensor in V. cholerae. The ToxRS complex exposes a bile binding pocket inside ToxS lipocalin-like barrel that is only properly built via stabilization by newly formed structural elements of ToxR. We performed multiple interaction experiments combined with extensive molecular dynamic MD simulations, to eventually present a bile binding ToxRSp complex, revealing contributions from both proteins to the interaction with bile acid. ToxRSp shows structural and functional similarities with bile sensing complex VtrAC from V. parahaemolyticus (Alnouti, 2009; Li et al., 2016; Tomchick et al., 2016) thus supporting a common superfamily of co-component signal transduction systems whose sensory function is strictly connected to an obligate heterodimer formation (Kinch et al., 2022). AlphaFold-Multimer (Evans et al., 2021; Jumper et al., 2021) structure predictions furthermore reveal a conserved fold of ToxS in different Vibrio species, in contrast to ToxR exhibiting a structural variability among different Vibrio strains. Results Periplasmic domains of V. cholerae ToxRS form an obligate dimer The crystal structure of ToxRp and ToxSp reveals the formation of a heterodimer, with ToxRp contributing secondary structure elements to ToxSp otherwise unstable ß-barrel fold (Figure 1, Figure 2, pdb: 8ALO). The complex forms spontaneously upon addition of both proteins. Figure 1 with 3 supplements see all Download asset Open asset Heterodimer formation of ToxRSp (pdb: 8ALO). ToxRp ß strand 5 completes main chain hydrogen bond network of ToxSp ß-barrel formation, by interaction with ToxSp ß1. ToxSp ß8 position is stabilized by main chain hydrogen bonds with ToxSp ß1, as well as side chain interactions with ToxRp ß5 and ß4. ToxRp ß5 and α2 are formed upon ToxSp interaction. ToxRSp crystal structure was determined by molecular replacement using an AlphaFold (Evans et al., 2021; Jumper et al., 2021) model as template. Figure 2 Download asset Open asset Detailed description of ToxRSp interface. (A) Overview of ToxRSp interface. The interaction is mainly established via ToxRp ß strands 3, 4 and 5, and ToxSp ß strands 6, 7 and 8. Additionally, an intermolecular salt bridge is formed between ToxRp R281 and ToxSp D142 located at the opening of ToxSp barrel. (B) The intermolecular main chain H-bond pattern of ToxRSp. The interprotein H-bond network is established between ToxRp ß5 and ToxSp ß1 thereby filling the gap between ToxSp ß1 and ß8, which interact only in the C-terminal region of the strands. ToxRp in the complex has an αß fold which is formed by an α-helix flanked by an anti-parallel hairpin and a three-stranded anti-parallel ß-sheet, followed by a C-terminal helix (Figure 1). Together, the ß-strands form a five-stranded anti-parallel ß-sheet. The C-terminal Cys293ToxRp is involved in a solution-oriented disulphide bond with Cys236ToxRp of the N-terminal helix (Figure 3). Figure 3 Download asset Open asset Comparison of ToxRp NMR structure (salmon) to ToxRp bound to ToxSp (green). Upon binding to ToxSp, ToxRp intrinsically disordered C-terminal region forms ß5 and α2. (A) Topology diagram of ToxRp and ToxRp bound to ToxSp. Newly formed structural elements are highlighted in grey. (B) Structural alignment between ToxRp (PDB 7NN6) and ToxRp in complex with ToxSp (PDB 8ALO). The C-terminal disulphide bond is shown in sticks. ToxSp forms a lipocalin-like fold consisting of an eight-stranded ß-barrel stabilized by intermolecular H-bond network and flanked by two α-helices located at the openings of the barrel (Figure 2, Supplementary file 2). For a correct barrel formation ToxSp ß1 and ß8 would need to be close enough for building main chain interactions. However, ToxSp ß8 contacts only the first three residues of nine residues long ToxSp ß1 (Figure 2). ToxRp ß5 is positioned into this gap and forms main chain H-bonds with remaining ToxSp ß1 residues. Additionally, close side-chain interactions between ToxRp ß4 and ToxSp ß8 further stabilize ToxSp ß8 positioning (Figure 2). ToxRp ß4 and ß3 are also involved in side-chain interactions with ToxSp ß7 and ß6 completing the strong complex formation. The residue Arg281 located in ToxRp ß5 forms an intermolecular salt bridge with Asp142 of ToxSp α2, which restricts the opening of the barrel (Figure 2). DSSP secondary structure analysis (Kabsch and Sander, 1983; Touw et al., 2015) indicates a ten-stranded intermolecular ß-sheet consisting of ß1-ß5 of ToxSp and ß1-ß5 of ToxRp. ToxSp five stranded ß-sheet (ß1-ß5) and three-stranded ß-sheet (ß6-ß8), result in a ß-barrel formation, which is stabilized mainly via hydrophobic core interactions and interactions with ToxRp ß5 (Figure 1). Mutational studies reveal that ToxSp mutant L33S results in increased proteolysis of ToxRp indicating a loss of function of ToxS (Pfau and Taylor, 1998). The crystal structure of ToxRSp shows that L33 is located at ToxSp α1 at the N-terminal membrane-oriented opening of the barrel of ToxSp (Figure 1—figure supplement 2). L33 forms central hydrophobic interactions with apolar residues of ToxSp ß strands stabilizing the core of the barrel (Figure 1—figure supplement 2.). A serine to leucine mutation probably disrupts the hydrophobic interactions and may result in a loss of structure, thus explaining the inability of ToxSp L33S mutant to protect ToxR from proteolysis (Pfau and Taylor, 1998). There are currently no structures of proteins with significant sequence homology to the ToxRSp complex available in the protein data bank. However, despite the lack of sequence identity the VtrAC complex of V. parahaemolyticus (Tomchick et al., 2016; pdb code: 5KEV, 5KEW) has striking structural and functional similarities with ToxRSp from V. cholerae (Figure 1—figure supplement 3, all-atom RMSD 5.348 Å calculated for 836 atoms; sequence identity: ToxRp-VtrA: 8.1%; ToxSp-VtrC: 11.7%). Similar to ToxRS, the bile sensing functionality of VtrAC induces an essential step for virulence activation upon human gastro-intestinal infection. VtrAC activates the production of main virulence factor VtrB which enables the production of a type III secretion system 2 (T3SS2) for the injection of virulence factors into host cells (Kaval et al., 2023; Zou et al., 2023). Intrinsically disordered ToxRp C-terminus folds into structural elements essential for the complex formation with ToxSp Small-angle X-ray scattering (SAXS) experiments indicate a dimer formation of ToxSp in the absence of ToxRp (Figure 6—figure supplement 1, Figure 6—figure supplement 2, Supplementary file 1c) supporting the recently proposed HDOCK ToxS-ToxS dimer (Canals et al., 2023). Nevertheless, aggregation of ToxSp in solution occurs rapidly suggesting an unstable dimer formation (Gubensäk et al., 2021b; Figure 6—figure supplement 3). The instability of ToxSp can be explained by the inability of the first and the last ß strand of the barrel to form strong hydrogen bonding, thus resulting in an unstable hydrophobic core and consequently aggregation. Interaction with ToxRp likely protects otherwise exposed hydrophobic regions of ToxSp (Figure 1, Figure 2). Comparison of the ToxRSp crystal structure with our previously determined NMR structure of ToxRp (pdb: 7NN6) (Gubensäk et al., 2021b), shows the formation of additional secondary structure elements upon complex formation (Figure 3). Unbound ToxRp has a long intrinsically disordered C-terminus in solution, containing the second cysteine Cys293ToxRp forming a disulphide bond with Cys236 ToxRp located in the middle of helix 1 (Gubensäk et al., 2021b). When bound to ToxSp, the unstructured region of the C-terminus of ToxRp performs a disorder to order transition, by forming two additional structural elements: short α helix 2 and ß strand 5 (Figure 3). The newly formed short α helix 2 contacts the turn between ToxSp ß1 and ß2, as well as residues Leu45ToxSp and Ile46ToxSp located at the C-terminal part of ToxSp ß1 thus increasing the interaction interface and further stabilizing the fold. Although the C-terminal region undergoes extreme structural changes upon complex formation, the orientation of the disulphide bond does not change drastically. ToxRp ß strand 5 is only formed upon complex formation with ToxSp and forms the essential basis for the interaction by building a stabilizing hydrogen bonding network with ß strands 1 and 8 of ToxSp (Figure 2). Newly formed ToxRp ß5 also forms hydrogen bonds with ToxRp ß4. In unbound ToxRp, polar residues of ß4 are pointing towards solution, whereas hydrophobic parts contribute to the hydrophobic core. Nevertheless, ToxRp ß4 does not reveal significant conformational changes upon ToxRSp complex formation. ToxSp in complex with ToxRp contains a bile binding pocket ToxRSp bile interaction was tested using NMR (Becker et al., 2018) (saturation transfer difference STD Figure 6—figure supplement 3, Figure 6—figure supplement 4), chemical shift perturbation CSP (Figure 7, Figure 6—figure supplement 5) and diffusion ordered spectroscopy DOSY (Figure 6B, Figure 6—figure supplement 6), as well microfluidic modulation spectroscopy MMS (Figure 4, Figure 4—figure supplement 1), size exclusion chromatography (Figure 6C) and SAXS (Figure 6A, Figure 6—figure supplement 1, Figure 6—figure supplement 2, Supplementary file 1c). Interaction experiments were performed with bile acid sodium cholate hydrate S-CH. All mentioned experiments confirm an interaction of the ToxRSp complex with bile. Figure 4 with 1 supplement see all Download asset Open asset Analysis of microfluidic modulation spectroscopy MMS experiments. MMS experiments of ToxRSp with different S-CH concentrations show conformational changes upon binding of bile to ToxRSp. Additional structural rearrangements could be detected upon higher additions of bile. (A) Bar chart of the relative abundance of secondary structural motifs based on Gaussian deconvolution of the corresponding spectra. (B) MMS spectra (baselined, inverted 2nd Derivative) of ToxRSp with and without bile, showing spectral changes upon higher bile additions. Figure 4—source data 1 MMS data. https://cdn.elifesciences.org/articles/88721/elife-88721-fig4-data1-v2.zip Download elife-88721-fig4-data1-v2.zip The lipocalin-like fold of ToxSp forms a hydrophobic cavity (Figure 5, Figure 5—figure supplement 1), similar to the bile binding pocket of VtrC in complex with VtrA from V. parahaemolyticus (Tomchick et al., 2016). Using extensive MD simulations enabled a detailed analysis of the protein-ligand interface. Upon binding of bile acid mainly loop regions undergo major conformational changes (Figure 4, Figure 5). Comparison of ToxRSp apo and bile-bound state reveals that ToxSp β1/β2 loop and ToxRp α1/β2 loop experience strongest rearrangements, whereas loop β3/β4ToxSp, α1ToxSp, α2ToxSp and loop β3/β4ToxRp reveal only minor structural changes (Figure 5C). Similarly, MMS spectra show significant spectral changes in disordered, turn and helical regions (Figure 4). Figure 5 with 1 supplement see all Download asset Open asset ToxRSp binding pocket for bile acid. (A) Superimposition of 100 structures of the ToxRSp complex (dark grey/light grey cartoons) with cholate (sticks, C-atoms in yellow) along one representative classical MD simulation. (B) Detailed view of the binding mode of cholate to ToxRSp. The residues with the larger contribution to the binding of the ligand are highlighted as well as residues 46–49 of ToxSp located on loop β1/β2. (C) Overlay of ToxRSp apo and bile-bound structures. Conformational changes mainly occur in ToxSp loop β1/β2 and ToxRp loop α1/β2. ToxSp helices α1 and α2 and ToxRp loop β3/β4, both close to the ToxRSp interface, experience minor conformational rearrangements upon bile interaction. (D) Hydrophobic properties of the bile binding cavity calculated with the CavMan (v. 0.1, 2019, Innophore GmbH plugin in PyMOL). The entrance of the cavity (blue to green) faces the solvent exhibiting a hydrophilic environment, in contrast to the buried hydrophobic areas (yellow to red). For the analysis of the hydrophobicity of the cavities the program VASCo (Steinkellner et al., 2009) was used; cavities were calculated using a LIGSITE algorithm (Hendlich et al., 1997). Figure 5—source data 1 ToxRSp MD data. https://cdn.elifesciences.org/articles/88721/elife-88721-fig5-data1-v2.zip Download elife-88721-fig5-data1-v2.zip The nature of the interactions of bile acid with the protein complex are mostly hydrophobic. The carboxylic moiety forms a strong interaction with Arg52ToxSp. Additionally, cholate interacts with residues Ser44ToxSp, Ile135ToxSp, Leu138ToxSp, Phe139ToxSp, Val283ToxRp, and Arg281ToxRp (Figure 5). Both proteins contribute to the bile acid interaction, although ToxSp residues are forming a major part of the interface. ToxRSp has additional surface exposed bile binding patches ToxRSp interaction experiments propose an increase of molecular weight upon bile addition as shown in Figure 6. SEC-SAXS experiments indicate an increase of molecular weight, radius of gyration and effective maximum particle dimension of ToxRSp upon addition of equimolar amounts of bile acid (Figure 6A, Figure 6—figure supplement 1, Figure 6—figure supplement 2, Supplementary file 1c). Similar outcomes are observed by SEC runs of ToxRSp with and without bile acid using a Superdex200 column resulting in a broadening of ToxRSp-bile acid peak and slightly decreased retention times (Figure 6C, Figure 6—figure supplement 7). NMR DOSY experiments of ToxRSp bound to bile acid clearly show a decrease of the diffusion coefficient, which is linked to an increase of size and weight (Figure 6B, Figure 6—figure supplement 5). Via MMS experiments conformational changes could be detected upon bile acid binding but clearly indicate that no severe structural rearrangements for example aggregation events occur (Figure 4, Figure 4—figure supplement 1). Figure 6 with 8 supplements see all Download asset Open asset ToxRSp bile interaction experiments propose additional bile interaction areas I-IV. (A) SEC-SAXS experiments with ToxRSp and S-CH reveal an increase of the radius of gyration RG and the effective maximum particle dimension Dmax upon bile addition. (B) Superimposed NMR DOSY spectra of ToxRSp with and without bile acid S-CH. Addition of bile acid causes a decrease of the diffusion coefficient due to an increase of molecular weight. (C) SEC experiments with ToxRSp and S-CH reveal a slight decrease of retention volume upon bile acid presence from 16.19 ml to to 16.08 ml and a broadening of the peak. (D) MD determined surface exposed bile interacting regions of ToxRSp. Besides the bile binding cavity of ToxSp, four regions (marked as I-IV) on the ToxRSp surface could be mapped as additional bile binding areas. Region I and III involves both proteins, whereas region II is located on ToxSp and region IV on ToxRp. The representative structure of the ToxRSp complex (ToxRp: dark grey, ToxSp: light grey) is shown with cholate bound at the cavity (orange) and additional cholate molecules attach to surface exposed regions of ToxRSp. Figure 6—source data 1 SEC data. https://cdn.elifesciences.org/articles/88721/elife-88721-fig6-data1-v2.zip Download elife-88721-fig6-data1-v2.zip Due to a loss of signals upon equimolar additions of bile acid caused by the previously mentioned increase of molecular weight, NMR titration experiments were performed using small additions of S-CH (Figure 7). Spectra were recorded with ToxRp and ToxSp both 15N isotopically labelled. An overlay of spectra of only ToxRp or ToxSp 15N labelled enabled the ascription of signals to each of the proteins (Figure 7A). Even small additions of bile (0.1 molar ratio) to ToxRSp results in a significant decrease of signal intensity. ToxSp seems to be mostly affected by the addition of bile acid, causing a disappearance of ToxSp signals indicating direct interaction events (Figure 7B–D). ToxRp signals are also affected by decreased signal intensities but to a lower extent when compared to ToxSp. Upon bile additions higher than a molar ratio of 0.6, mostly signals from ToxRp are visible (Figure 7D). In general, all ToxRSp peaks experience decreased signal intensities caused by fast T2 relaxation. Figure 7 Download asset Open asset NMR titration of S-CH to 15 N labelled ToxRSp. (A) Overlay of 15N-HSQC spectra of 15N-labelled ToxRSp (purple) with 15N-labelled ToxRp (green) and ToxSp (blue) for ascription of peaks to each of the proteins. (B–D) Overlay of 15N-HSQC spectra of 15N-labelled ToxRSp before (purple) and after the addition of increasing ratios of S-CH (orange): 0.3 (B), 0.5 (C), 0.7 (D). Upon bile acid addition signal intensities significantly decrease. Upon higher additions of bile acid mainly ToxRp peaks are visible indicated by green asterisk in (D), whereas most ToxSp signals disappear indicating direct interaction with S-CH. Remaining ToxSp signals are marked with blue asterisk. Figure 7—source data 1 NMR titration 1 ToxRSp plus S-CH. https://cdn.elifesciences.org/articles/88721/elife-88721-fig7-data1-v2.zip Download elife-88721-fig7-data1-v2.zip Figure 7—source data 2 NMR titration 2 ToxRSp plus S-CH. https://cdn.elifesciences.org/articles/88721/elife-88721-fig7-data2-v2.zip Download elife-88721-fig7-data2-v2.zip Interestingly, MMS experiments show additional structural changes of ToxRSp upon higher bile acid concentrations, with maximum changes achieved at a double molar excess of bile acid suggesting the presence of additional binding sites on ToxRSp (Figure 4, Figure 4—figure supplement 1). Prompted by this evidences, further MD simulations of the complex ToxRSp were performed with one cholate molecule at the binding cavity in the presence of 14 additional cholate molecules randomly positioned in the solvent (Figure 6D). Four solvent exposed regions of the protein, which show a transient binding of cholate molecules could be identified (Figure 6D). Three are located on ToxSp, of which two implicate interactions with ToxRp. One interaction site is exclusively located on ToxRp. These results support the experimental finding of an increase of the molecular weight by binding of more than one cholate molecule. The active state of ToxR is proposed to be dimeric due to its binding to direct repeat DNA sequences (Canals et al., 2023; Gubensäk et al., 2021a; Krukonis and DiRita, 2003; Withey and DiRita, 2006). Nevertheless, the increase of molecular weight observed in the interaction experiments is significantly lower than 30 kDa (Figure 6A–C), which corresponds to the molecular weight of ToxRSp. Interaction experiments therefore do not implement a bile induced dimerization or oligomerization event. A bile induced formation of trimers involving two ToxRp and one ToxSp or two ToxSp and one ToxRp molecules also seems unlikely. A described trimer formation would involve the disruption of ToxRSp heterodimer complexes, which exhibits a dissociation constant at nanomolar range (Gubensäk et al., 2021b), and includes aggregation of unstable ToxSp. Also, MMS experiments reveal subtle conformational changes of ToxRSp upon bile interaction and do not support extreme structural rearrangements. Instead of major structural changes like dimerization or loss of structure due to aggregation, the experimental results more likely indicate that the presence of bile leads to local conformational changes near the binding cavity of the ToxRSp barrel, similar to the VtrAC complex of V. parahaemolyticus (Li et al., 2016; Tomchick et al., 2016). Taken together, we propose that ToxRSp bile sensing functions in a complex manner involving a binding site in ToxRSp barrel as well as surface exposed binding regions for bile acid. Bile acid interaction results in concentration dependent conformational changes of ToxRSp which may be related to ToxR transcriptional activity changes (Eichinger et al., 2011; Gubensäk et al., 2021a; Kenney, 2002; Martínez-Hackert and Stock, 1997a; Martínez-Hackert and Stock, 1997b). ToxRp ß-sheet forms a low-affinity binding region for bile The MD determined ToxRp binding region to bile (Figure 6D) is also confirmed experimentally via chemical shift mapping (Figure 8, Figure 8—figure supplement 1; Becker et al., 2018; Williamson, 2013). The bile binding area is located at the solution-oriented region of the ß-sheet of ToxRp. The determined dissociation constant of 2.6±1.42 mM

The seventh pandemic of the diarrheal cholera disease, which began in 1960, is caused by the Gram-negative bacterium Vibrio cholerae. Its environmental persistence provoking recurring sudden outbreaks is enabled by V. cholerae's rapid adaption to changing environments involving sensory proteins like ToxR and ToxS. Located at the inner membrane, ToxR and ToxS react to environmental stimuli like bile acid, thereby inducing survival strategies for example bile resistance and virulence regulation. The presented crystal structure of the sensory domains of ToxR and ToxS in combination with multiple bile acid interaction studies, reveals that a bile binding pocket of ToxS is only properly folded upon binding to ToxR. Our data proposes an interdependent functionality between ToxR transcriptional activity and ToxS sensory function. These findings support the previously suggested link between ToxRS and VtrAC-like co-component systems. Besides VtrAC, ToxRS is now the only experimentally determined structure within this recently defined superfamily, further emphasizing its significance. In-depth analysis of the ToxRS complex reveals its remarkable conservation across various Vibrio species, underlining the significance of conserved residues in the ToxS barrel and the more diverse ToxR sensory domain. Unravelling the intricate mechanisms governing ToxRS's environmental sensing capabilities, provides a promising tool for disruption of this vital interaction, ultimately inhibiting Vibrio's survival and virulence. Our findings hold far-reaching implications for all Vibrio strains that rely on the ToxRS system as a shared sensory cornerstone for adapting to their surroundings.

The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world.

In late 2020, the results of CASP14, the 14th event in a series of competitions to assess the latest developments in computational protein structure-prediction methodology, revealed the giant leap forward that had been made by Google's Deepmind in tackling the prediction problem. The level of accuracy in their predictions was the first instance of a competitor achieving a global distance test score of better than 90 across all categories of difficulty. This achievement represents both a challenge and an opportunity for the field of experimental structural biology. For structure determination by macromolecular X-ray crystallography, access to highly accurate structure predictions is of great benefit, particularly when it comes to solving the phase problem. Here, details of new utilities and enhanced applications in the CCP4 suite, designed to allow users to exploit predicted models in determining macromolecular structures from X-ray diffraction data, are presented. The focus is mainly on applications that can be used to solve the phase problem through molecular replacement.

XDSGUI is a lightweight graphical user interface (GUI) for the XDS, SHELX and ARCIMBOLDO program packages that serves both novice and experienced users in obtaining optimal processing and phasing results for X-ray, neutron and electron diffraction data. The design of the program enables data processing and phasing without command line usage, and supports advanced command flows in a simple user-modifiable and user-extensible way. The GUI supplies graphical information based on the tabular log output of the programs, which is more intuitive, comprehensible and efficient than text output can be.