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Oss, Netherlands

Harvey B.H.,North West University South Africa | Shahid M.,MSD
Pharmacology Biochemistry and Behavior | Year: 2012

Anxiety disorders are amongst the most common and disabling of psychiatric illnesses and have severe health and socio-economic implications. Despite the availability of a number of treatment options there is still a strong medical need for novel and improved pharmacological approaches in treating these disorders. New developments at the forefront of preclinical research have begun to identify the therapeutic potential of molecular entities integral to the biological response to adversity, particularly molecules and processes that may pre-determine vulnerability or resilience, and those that may act to switch off or "unlearn" a response to an aversive event. The glutamate system is an interesting target in this respect, especially given the impact anxiety disorders have on neuroplasticity, cognition and affective function. These areas of research demonstrate expanding and improved evidence-based options for treating disorders where stress in various guises plays an important etiological role. The current review will discuss how these pathways are involved in fear circuitry of the brain and compare the strength of therapeutic rationale as well as progress towards pharmacological validation of the glutamate pathway towards the treatment of anxiety disorders, with a particular focus on metabotropic and ionotropic glutamate receptors. Specific reference to their anxiolytic actions and efficacy in translational disease models of posttraumatic stress disorder, obsessive-compulsive disorder, panic disorder and phobia will be made. In addition, the availability of ligands necessary to assist clinical proof of concept studies will be discussed. © 2011 Elsevier Inc. All rights reserved.

Kerr W.J.,University of Strathclyde | Morrison A.J.,MSD | Pazicky M.,University of Strathclyde | Weber T.,University of Strathclyde
Organic Letters | Year: 2012

Bismesitylmagnesium has been shown to successfully mediate the Shapiro reaction. A range of tosylhydrazones has been subjected to the developed system, which furnishes exceptionally high incorporation of the introduced electrophiles and good yields of the functionalized styrenes. At conveniently accessible temperatures and with a comparably small excess of base reagent, this protocol offers an efficient alternative to the lithium-mediated process. Importantly, 1.05 equiv of Weinreb amides are sufficient to obtain aryl enones in good yields. © 2012 American Chemical Society.

Harker W.R.R.,University of Bath | Carswell E.L.,MSD | Carbery D.R.,University of Bath
Organic Letters | Year: 2010

The E/Z-selectivity in the formation of silylketene acetals derived from phenylacetate esters, mediated by LiHMDS, has been studied by in situ NMR techniques. The formation is seen to be highly E-selective with use of the newly developed protocol. Isolated aryl-substituted silylketene acetals are now attainable with high levels of E-geometrical purity in excellent yield. © 2010 American Chemical Society.

Gerrits M.,MSD | Mannaerts B.,MSD | Kramer H.,MSD | Hanssen R.,MSD
Journal of Clinical Endocrinology and Metabolism | Year: 2013

Context: Two new low-molecular-weight LH agonists (Org 43553 and Org 43902) were shown to induce ovulation in preclinical experiments. Objective: Our objective was to assess the safety, pharmacokinetics, and pharmacodynamics of Org 43553 and Org 43902 when administered to healthy females. Design and Setting: Org 43553 and 43902 studies were randomized, placebo-controlled, singlerising-dose first-in-human trials, which included 159 healthy female volunteers. Part 1 of the studies assessed the safety and pharmacokinetics. Part 2 evaluated the pharmacodynamics effect of a single oral dose of Org 43553 (25-900 mg) or Org 43902 (30-300 mg) to induce ovulation afterthe development of a large preovulatory follicle, whereas the endogenous LH surge was suppressed due to GnRH antagonist treatment while follicular development was supported with recombinant FSH. Results: Org 43553 and 43902 were safe and well tolerated. Both compounds showed a fast absorption after oral intake, with peak concentrations reached within 0.5 to 1 hour. The elimination half-life of Org 43553 was 30 to 47 hours and that of Org 43902 was 17 to 22 hours. Ovulation induction confirmed by midluteal progesterone rise ≥15 nmol/L was proven in both studies, also when excluding subjects with an endogenous LH rise. The minimal effective dose for ovulation induction was 300 mg in both studies and resulted in an ovulation rate of 83% and 82%, respectively. Conclusions: These first proof-of-concept studies both demonstrated that a single oral intake of an low-molecular-weight LH agonist induces ovulation of the preovulatory follicle in pituitary-suppressed female volunteers of reproductive age. Copyright © 2013 by The Endocrine Society.

No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. The strains, plasmids and oligonucleotides used in this study are listed in the Supplementary Table. The enteroaggregative E. coli EAEC strain 17-2 and its ∆tssA, ∆tssBC, ∆tssE, ∆tssF, ∆tssG, ∆tssK, ∆tssL, ∆tssM, ∆hcp, ∆vgrG and tssB-mCherry isogenic derivatives were used for this study11, 15, 25. The E. coli K-12 DH5α, W3110, BTH101 and T7 Iq pLys strains were used for cloning steps, co-immunoprecipitation, bacterial two-hybrid and protein purification, respectively. Strains were routinely grown in LB rich medium (or Terrific broth medium for protein purification) or in Sci-1 inducing medium (SIM; M9 minimal medium, glycerol 0.2%, vitamin B1 1 μg ml−1, casaminoacids 100 μg ml−1, LB 10%, supplemented or not with bactoagar 1.5%) with shaking at 37 °C31. Plasmids were maintained by the addition of ampicillin (100 μg ml−1 for E. coli K-12, 200 μg ml−1 for EAEC), kanamycin (50 μg ml−1) or chloramphenicol (30 μg ml−1). Expression of genes from pBAD, pETG20A/pRSF or pASK-IBA vectors was induced at A   ≈ 0.6 with 0.02% of l-arabinose (Sigma-Aldrich) for 45 min, 0.5–1 mM of isopropyl-β-d-thio-galactopyrannoside (IPTG, Eurobio) for 14 h or 0.02 μg ml−1 of anhydrotetracyclin (AHT, IBA Technologies) for 45 min, respectively. For BACTH experiments, plates were supplemented with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal, Eurobio, 40 μg ml−1). The tssA gene was deleted into the enteroaggregative E. coli 17-2 strain using a modified one-step inactivation procedure32 as previously described11 using plasmid pKOBEG33. In brief, a kanamycin cassette was amplified from plasmid pKD46 using oligonucleotides carrying 50-nucleotide extensions homologous to regions adjacent to tssA. After electroporation of 600 ng of column-purified PCR product, kanamycin-resistant clones were selected and verified by colony-PCR. The kanamycin cassette was then excised using plasmid pCP20 (ref. 32). The deletion of tssA was confirmed by colony-PCR. The same procedure was used to introduce the mCherry-coding sequence upstream the stop codon of the tssB gene (vector pmCh-KD4 as template for PCR amplification) or the sfGFP-coding sequence downstream the start codon (vector pKD4-sfGFP as template) or upstream the stop codon (vector psfGFP-KD4 as template) of the tssA gene to yield strains producing TssB–mCherry, sfGFP–TssA or TssA–sfGFP from their original chromosomal loci. All bacterial two-hybrid plasmids and the plasmid producing the TssJLM membrane core complex (pRSF-TssJST-FLTssL-6HisTssM, pRSF-TssJLM) have been described previously13, 25. PCR was performed using a Biometra thermocycler using the Q5 (New England Biolabs) or Pfu Turbo (Agilent Technologies) DNA polymerases. Restriction enzymes were purchased from New England Biolabs and used according to the manufacturer’s instructions. Custom oligonucleotides were synthesized by Sigma Aldrich and are listed in the Supplementary Table. Enteroaggregative E. coli 17-2 chromosomal DNA was used as a template for all PCR. E. coli strain DH5α was used for cloning procedures. All the plasmids (except for pETG20A and pDEST17 derivatives) have been constructed by restriction-free cloning34 as previously described25. In brief, the gene of interest was amplified using oligonucleotides introducing extensions annealing to the target vector. The double-stranded product of the first PCR has then been used as oligonucleotides for a second PCR using the target vector as template. PCR products were then treated with DpnI to eliminate template plasmids and transformed into DH5α-competent cells. For protein purification, the sequences encoding the full-length TssA (residues 1–542), the TssA (residues 1–392), the TssA (residues 221–377) and TssA (residues 393–542) domains, the N-terminal domain of VgrG (residues 1– 490), the full-length TssE or both TssB and TssC were cloned into the pETG-20A (TssA, TssA , VgrG , TssE) or pDEST17 (TssA , TssBC) expression vector (gifts from A. Geerlof, EMBL, Hamburg) according to standard Gateway protocols. Proteins produced from pETG20A derivatives are fused to an N-terminal 6 × His-tagged thioredoxin (TRX) followed by a cleavage site for the Tobacco etch virus (TEV) protease whereas proteins produced from pDEST17 are fused to an N-terminal 6 × His tag followed by a TEV protease cleavage site. All constructs have been verified by restriction analyses and DNA sequencing (Eurofins or MWG). The adenylate cyclase-based bacterial two-hybrid technique35 was used as previously published36. In brief, the proteins to be tested were fused to the isolated T18 and T25 catalytic domains of the Bordetella adenylate cyclase. After introduction of the two plasmids producing the fusion proteins into the reporter BTH101 strain, plates were incubated at 30 °C for 48 h. Three independent colonies for each transformation were inoculated into 600 μl of LB medium supplemented with ampicillin, kanamycin and IPTG (0.5 mM). After overnight growth at 30 °C, 10 μl of each culture were dropped onto LB plates supplemented with ampicillin, kanamycin, IPTG and X-Gal and incubated for 16 h at 30 °C. The experiments were done at least in triplicate and a representative result is shown. Fluorescence microscopy experiments have been performed essentially as described13, 15, 21, 25. In brief, cells were grown overnight in LB medium and diluted to an A   ≈ 0.04 into Sci-1 inducing medium (SIM). Exponentially growing cells (A   ≈ 0.8–1) were harvested, washed in phosphate buffered saline buffer (PBS), resuspended in PBS to an A   ≈ 50, spotted on a thin pad of 1.5% agarose in PBS, covered with a cover slip and incubated for one hour at 37 °C before microscopy acquisition. For each experiment, ten independent fields were manually defined with a motorized stage (Prior Scientific) and stored (x, y, z, PFS-offset) in our custom automation system designed for time-lapse experiments. Fluorescence and phase contrast micrographs were captured every 30 s, using an automated and inverted epifluorescence microscope TE2000-E-PFS (Nikon, France) equipped with a perfect focus system (PFS). PFS automatically maintains focus so that the point of interest within a specimen is always kept in sharp focus at all times despite mechanical or thermal perturbations. Images were recorded with a CoolSNAP HQ 2 (Roper Scientific, Roper Scientific SARL, France) and a 100× /1.4 DLL objective. The excitation light was emitted by a 120 W metal halide light. All fluorescence images were acquired with a minimal exposure time to minimize bleaching and phototoxicity effects. The sfGFP images were recorded by using the ET-GFP filter set (Chroma 49002) using an exposure time of 200–400 ms. The mCherry images were recorded by using the ET-mCherry filter set (Chroma 49008) using an exposure time of 100–200 ms. Slight movements of the whole field during the time of the experiment were corrected by registering individual frames using StackReg and Image Stabilizer plugins for ImageJ. sfGFP and mCherry fluorescence channels were adjusted and merged using ImageJ (http://rsb.info.nih.gov/ij/). For statistical analyses, fluorescent foci were automatically detected. First, noise and background were reduced using the ‘Subtract Background’ (20 pixels Rolling Ball) plugin from Fiji37. The sfGFP foci were automatically detected by a simple image processing: (1) create a mask of cell surface and dilate; (2) detect the individual cells using the ‘Analyse particle’ plugin of Fiji; (3) sfGFP foci were identified by the ‘Find Maxima’ process in Fiji. To avoid false positives, each event was manually controlled in the original data. Box-and-whisker representations of the number of foci per cell were made with R software. t-tests were performed in R to statistically compare each population. Kymographs were obtained after background fluorescence substraction and sectioning using the Kymoreslicewide plug-in under Fiji37. Fluorescent foci were detected using a local and sub-pixel resolution maxima detection algorithm and tracked over time with a specifically developed plug-in for ImageJ. The x and y coordinates were obtained for each fluorescent focus on each frame. The mean square displacement (MSD) was calculated as the distance of the foci from its location at t = 0 at each time using R software and plotted over time. For each strain tested, the MSD of at least 10 individual focus trajectories was calculated. For statistical analyses of mobile trajectories, kymograph analyses were performed and the percentage of fixed, mobile with random dynamics and mobile with unidirectional trajectory foci are reported. FLIM experiments were carried on the same microscope device used for the time-lapse microscopy experiments except with a laser of 488 nm. For each cell a region of interest that corresponds to the size of the laser beam was focused away from TssB–mCherry sheath-labelled sfGFP–TssA for a time of 3 s at a maximum intensity of 100%. The extinction of the complete sfGFP–TssA pool was checked by (i) the absence of recovery of bleached sfGFP–TssA-membrane clusters and (ii) by the overall drop and lack of recovery in intracellular intensity. E. coli T7 Iq pLysS cells bearing pETG20A or pDEST17 derivatives were grown at 37 °C in Terrific Broth to an A   ≈ 0.9 and gene expression was inducted with 0.5 mM IPTG for 16 h at 17 °C. Cells were harvested, resuspended in Tris-HCl 20 mM pH 8.0, NaCl 150 mM and lysozyme (0.25 mg ml−1) and broken by sonication. Soluble proteins were separated from inclusion bodies and cell debris by centrifugation 30 min at 20,000g. The His-tagged fusions were purified using ion metal Ni2+ affinity chromatography (IMAC) using a 5-ml HisTrap column (GE Healthcare) and eluted with a step gradient of imidazole. The fusion proteins were further digested overnight at 4 °C by a hexahistidine-tagged TEV protease using a 1:10 (w/w) protease:protein ratio. The TEV protease and contaminants were retained by a second IMAC and the purified proteins were collected in the flow through. Proteins were further separated on preparative Superdex 200 or Superose 6 gel filtration column (GE Healthcare) equilibrated in Tris-HCl 20 mM pH 8.0, NaCl 150 mM. The fractions containing the purified protein were pooled and concentrated by ultrafiltration using the Amicon technology (Millipore, California, USA). The seleno-methionine (SeMet) derivatives of the N- and C-terminal domains of TssA were produced in minimal medium supplemented with 100 mg l−1 of lysine, phenylalanine and threonine, 50 mg l−1 of isoleucine, leucine, valine and seleno-methionine. Gene induction and protein purification were performed as described above. The full-length TssA protein was subjected to Proteinase K limited proteolysis (1:10 protease:protein ratio). The reaction was quenched at different time points by the addition of 1 mM PMSF and further boiling for 10 min at 96 °C. Samples were analysed by SDS–PAGE and Coomassie blue staining. Digested products were identified by Edman N-terminal sequencing and electrospray mass sprectrometry (Proteomic platform, Institut de Microbiologie de la Méditerranée, Marseille, France). Size-exclusion chromatography (SEC) was performed on an Alliance 2695 HPLC system (Waters) using KW803 and KW804 columns (Shodex) run in Tris-HCl 20 mM pH 8.0, NaCl 150 mM at 0.5 ml per min. MALS, UV spectrophotometry, QELS and RI were monitored with MiniDawn Treos (Wyatt Technology), a Photo Diode Array 2996 (Waters), a DynaPro (Wyatt Technology) and an Optilab rEX (Wyatt Technology), respectively, as described12. Mass and hydrodynamic radius calculation were performed with the ASTRA software (Wyatt Technology) using a dn/dc value of 0.185 ml g−1. Steady-state interactions were monitored using a BIAcore T200 at 25 °C12. All the buffers were filtered on 0.2 μm membranes before use. The HC200m sensor chip (Xantech) was coated with purified Hcp, VgrG, TssE or TssBC complex, immobilized by amine coupling (∆RU = 4,000–4,300). A control flow-cell was coated with thioredoxin immobilized by amine coupling at the same concentration (∆RU = 4,100). Purified TssA, TssA N-terminal and TssA C-terminal domains (six concentrations ranging from 3.125 to 100 μM) were injected and binding traces were recorded in duplicate. The signal from the control flow cell and the buffer response were subtracted from all measurements. The dissociation constants (K ) were estimated using the GraphPad Prism 5.0 software on the basis of the steady state levels of ∆RU, directly related to the concentration of the analytes. The K were estimated by plotting on x axis the different concentration of analytes and the different ∆RU at a fixed time (5 s before the end of the injection step) on the y axis. For K calculation, nonlinear regression fit for xy analysis was used and one site (specific binding) as a model which corresponds to the equation y = B  × x/(K + x). Different combinations of plasmids were transformed in BL21(DE3): (i) pRSF-TssJLM + pIBA37(+); (ii) pRSF + pIBA37-FLTssA; (iii) pRSF-TssJLM + pIBA37-FLTssA; and (iv) pRSF-TssJM + pIBA37-FLTssA. Transformed BL21(DE3) cells were grown at 37 °C in 200 ml LB medium supplemented with kanamycin and ampicillin until A    ≈ 0.6 and gene induction was achieved by the addition of IPTG (1 mM) and anhydrotetracycline (0.02 μg ml−1) during 15 h at 16 °C. After cell lysis through three passages at the French press, total membranes were isolated as described previously13. Membranes were solubilized by the addition of 1% Triton X-100 (Affimetrix). Solubilized membrane fractions were purified on a 1 ml Streptactin column (GE Healthcare). The column was washed with buffer S (HEPES 50 mM pH 7.5, NaCl 50 mM, Triton X-100 0.075%) and bound proteins were eluted with buffer S supplemented with desthiobiotin (2.5 mM) and visualized by Coomassie blue staining and immunoblotting. For electron microscopy (EM) analyses, BL21(DE3) cells producing TssJLM and Flag-tagged TssA were grown and the TssJLM-A complex was purified as described for the TssJLM membrane core complex13. After the two consecutive affinity columns (His- and Strep-Trap-HP), the pooled fractions were injected onto a Superose 6 10:300 column equilibrated in HEPES 50 mM pH 7.5, NaCl 50 mM supplemented with 0.025% DM-NPG. Nine microlitres of the purified TssJLMA complex (~ 0.01 mg ml−1) were incubated to glow-discharged carbon-coated copper grids (Agar Scientific) for 30 s. After absorption, the sample was blotted, washed with three drops of water and then stained with 2% uranyl acetate. Images were collected on an FEI Tecnai F20 FEG microscope operating at a voltage of 200 kV, equipped with a direct electron detector (Falcon II) at 50,000 magnification. Nine microlitres of the purified full-length TssA protein (~ 0.01 mg ml−1) were incubated on a glow-discharged carbon-coated copper grid (Agar Scientific) for 30 s. After absorption, the sample was blotted, washed with three drops of water and then stained with 2% uranyl acetate. Images were recorded automatically using the EPU software on a FEG microscope operating at a voltage of 200 kV and a defocus range of 0.6–25 nm, using a FEI Falcon-II detector (Gatan) at a nominal magnification of 50,000, yielding a pixel size of 1.9 Å. A dose rate of 25 electrons per Å2 per second, and an exposure time of 1 s were used. A total of 100,000 particles were automatically selected from 500 independent images and extracted within boxes of 180 pixels × 180 pixels using EMAN2/BOXER38. The CTF was estimated and corrected by phase flipping using EMAN2 (e2ctf). All two- and three-dimensional (2D and 3D) classifications and refinements were performed using RELION 1.3 (refs 39, 40). The automatically selected data set was cleaned up by three rounds of reference-free 2D class averaging, and highly populated classes displaying high-resolution features were conserved and a final data set of 20,000 particles was assembled. An initial 3D-model was generated in EMAN2 using using 30 classes. 3D classification was then performed in Relion with five classes. The particles corresponding to most populated class (~18,000) were used for refinement. The Relion auto-refine procedure was used to obtain a final reconstruction at ~19 Å resolution after masking and with D6 symmetry imposed. Reported resolutions are based on the gold-standard Fourier shell correlation (FSC) 0.143 criterion; the FSC curve was corrected for the effects of a soft mask on the FSC curve using high-resolution noise substitution (Extended Data Fig. 5o)41. All density maps were corrected for the modulation transfer function of the detector and then sharpened by applying a negative B-factor (−1000) that was estimated using automated procedures. The electron microscopy map of the EAEC TssA full-length protein has been deposited in the Electron Microscopy Data Bank under accession number EMD-3282. Small-angle X-ray scattering (SAXS) analyses were performed at the ID29 beamline (European Synchrotron Radiation Facility, Grenoble, France) at a working energy of 12.5 keV (λ = 0.931 Å). Thirty microlitres of protein solution at 1.6, 3.7, 7.1, 9.8 and 14.9 mg ml−1 in Tris-HCl 20 mM pH 8.0, NaCl 150 mM were loaded by a robotic system into a 2-mm quartz capillary mounted in a vacuum and ten independent 10-s exposures were collected on a Pilatus 6M-F detector placed at a distance of 2.85 m for each protein concentration. Individual frames were processed automatically and independently at the beamline by the data collection software (BsxCUBE), yielding radially averaged normalized intensities as a function of the momentum transfer q, with q = 4πsin(θ)/λ, where 2θ is the total scattering angle and λ is the X-ray wavelength. Data were collected in the range q = 0.04–6 nm−1. The ten frames were combined to give the average scattering curve for each measurement. Data points affected by aggregation, possibly induced by radiation damage, were excluded. Scattering from the buffer alone was also measured before and after each sample analysis and the average of these two buffer measures was used for background subtraction using the program PRIMUS42 from the ATSAS package43. PRIMUS was also used to perform Guinier analysis44 of the low q data, which provides an estimate of the radius of gyration (R ). Regularized indirect transforms of the scattering data were carried out with the program GNOM45 to obtain P(r) functions of interatomic distances. The P(r) function has a maximum at the most probable intermolecular distance and goes to zero at D , the maximum intramolecular distance. The values of D were chosen to fit with the experimental data and to have a positive P(r) function. Three-dimensionnal bead models that fit with the scattering data were built with the program DAMMIF46. Ten independent DAMMIF runs were performed using the scattering profile of TssA, with data extending up to 0.35 nm−1, using slow mode settings, assuming P6 symmetry and allowing for a maximum 500 steps to grant convergence. The models resulting from independent runs were superimposed using the DAMAVER suite47 yielding an initial alignment of structures based on their axes of inertia followed by minimisation of the normalized spatial discrepancy (NSD)48. The NSD was therefore computed between a set of ten independent reconstructions, with a range of NSD from 0.678 to 0.815. The aligned structures were then averaged, giving an effective occupancy to each voxel in the model, and filtered at half-maximal occupancy to produce models of the appropriate volume that were used for all subsequent analyses. All the models were similar in terms of agreement with the experimental data, as measured by DAMMIF χ parameter and the quality of the fit to the experimental curve (calculated ). The SAXS data parameters are provided in Extended Data Table 1. Seleno-methionine (SeMet)-labelled TssA and TssA crystallization trials were carried out by the sitting-drop vapour diffusion method in 96-well Greiner crystallization plates at 20 °C, using a nanodrop-dispensing robot (Cartesian Inc.). Crystals of SeMet-labelled TssA grew in a few days after mixing 300 nl of protein at 4.7 mg ml−1 with 100 nl of 20% PEG 8000, 0.2 M calcium acetate, 0.1 M MES pH 6.8. Crystals of SeMet-labelled TssA grew in a few days after mixing 300 nl of protein at 4.7 mg ml−1 with 100 nl of 29% PEG 3350, 0.1 M HEPES pH 7.5. Crystals were cryoprotected with mother liquor supplemented with 20% polyethylene glycol and flash frozen in liquid nitrogen. Data sets were collected at the SOLEIL Proxima 1 beamline (Saint-Aubin, France). After processing the data with XDS49, the scaling was performed with SCALA and the structures were solved using the SHELXD program50. The structure was refined with AutoBUSTER51 alternated with model rebuilding using COOT52. The final data collection and refinement statistics are provided in Extended Data Table 2. The Ramachadran plots of the TssA and TssA structures exhibit 90.7/3.3 and 91.8/2.9 residues in the favoured and outlier areas, respectively. Figures were made with PyMOL53. The tail sheath modelling was performed using the Vibrio cholerae VipAB (TssBC) complex as starting structure23 (PDB: 3J9G) and the contracted tail sheath structures of Vibrio cholerae23. To date, however, the molecular structure of the extended (non-contracted) sheath is not available. In a recent paper, a low-resolution model of the extended VipAB sheath was modelled using the low-resolution EM map of the extended T4 phage tail sheath22. By superimposing the VipAB EM map to the gp18 bacteriophage T4 sheath protein structure, gross features of the sheath structure were obtained22. A similar approach was applied with Chimera54 using the VipAB molecular model in the extended T4 phage tail sheath instead of using the low-resolution VipAB EM map, yielding a model similar to that of Kube et al.22, but with molecular details. The sheath internal channel diameter shrinks from 110 to ~95 Å, and the external diameter from ~290 Å to ~190 Å. The internal diameter of the tail sheath makes it possible to fit stacked Hcp hexamers that are in contact with the tail sheath internal wall. Both extended and contracted tail sheath conformations were used to explore the faisability of sheath complexes with TssA using its EM map. TssA being at the distal end of the sheath, the polarity of the sheath was taken into consideration. It was suggested that the polarity of T6SS tail sheath is similar to that of bacteriophage T4 and therefore that the VipA (TssB) N-terminal and VipB (TssC) C-terminal helices point to and contact the baseplate31. TssA was therefore docked at the opposite extremity of the tail sheath using Chimera54. Hcp release11, 25 and fractionation assays11, 19, 25 have been performed as previously described. SDS-polyacrylamide gel electrophoresis was performed using standard protocols. For immunostaining, proteins were transferred onto 0.2-μm nitrocellulose membranes (Amersham Protran), and immunoblots were probed with primary antibodies, and goat secondary antibodies coupled to alkaline phosphatase, and developed in alkaline buffer with 5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium. The anti-TolB polyclonal antibodies are from our laboratory collection, while the anti-Flag (M2 clone, Sigma Aldrich) and anti-EFTu (Roche) monoclonal antibodies and alkaline phosphatase-conjugated goat anti-rabbit or anti-mouse secondary antibodies (Beckman Coulter) have been purchased as indicated. Coordinates and structure factors have been deposited in the Protein Data Bank under accession numbers 4YO3 and 4YO5 for TssA and TssA , respectively. Electron microscopy map for full-length TssA has been deposited in the Electron Microscopy Databank (EMDB) under accession code EMD-3282.

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