Computational protocol: An intermolecular binding mechanism involving multiple LysM domains mediates carbohydrate recognition by an endopeptidase

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[…] The full-length protein structure was solved using selenomethionine-derivative crystals as described previously (Wong & Blaise, 2013). Briefly, the selenomethionine-derivative protein was crystallized at 19°C in hanging drops composed of 1 µl protein solution at 24 mg ml−1 and 1 µl reservoir solution consisting of 0.1 M sodium citrate pH 5.5, 16%(w/v) PEG 4000, 15%(v/v) 2-propanol equilibrated against 500 µl reservoir solution. Crystals were soaked briefly in cryoprotectant solution consisting of 0.1 M sodium citrate pH 5.5, 16%(w/v) PEG 4000 and 20% ethylene glycol prior to being cryocooled in liquid nitrogen. Data collection was performed at a wavelength of 0.978 Å on the I911-3 beamline at the MAX-lab synchrotron, Lund, Sweden (Ursby et al., 2013) as described previously (Wong & Blaise, 2013). The structure was solved by single-wavelength anomalous dispersion (SAD) phasing (Hendrickson & Teeter, 1981) as described in Wong & Blaise (2013).P60_2LysM was crystallized at 19°C in conditions consisting of 28%(w/v) PEG MME 2000 and 0.1 M potassium thio­cyanate. Sitting drops set up by adding 1 µl reservoir solution to 1 µl 50 mg ml−1 protein solution were equilibrated against 500 µl reservoir solution. The crystal was soaked briefly in mother liquor containing 34%(w/v) PEG MME 2000 prior to cryocooling in liquid nitrogen. Data were collected on the I911-3 beamline at MAX-lab. The data set consisted of 200 frames collected with 1° oscillation range, 5 s exposure time, a wavelength of 0.98 Å and a crystal-to-detector distance of 204.8 mm.The structure of P60_2LysM bound to chitohexaose was obtained by co-crystallizing the two LysM domains with chitohexaose (Megazyme) at a protein:sugar molar ratio of 1:2 by dissolving the carbohydrate powder directly in the protein solution and incubating it overnight on ice. The complex was crystallized at 19°C in conditions consisting of 1.6 M ammonium sulfate, 0.1 M MES pH 6.5 and 5%(v/v) 1,4-dioxane. Hanging drops set up by adding 0.5 µl reservoir solution to 0.5 µl protein solution at 32 mg ml−1 were equilibrated against 500 µl reservoir solution. The crystal was soaked briefly in a solution consisting of 1.6 M ammonium sulfate, 0.1 M MES pH 6.5, 20%(v/v) 1,4-dioxane and 5%(v/v) glycerol prior to cryocooling in liquid nitrogen. Data were collected on the I911-2 beamline at MAX-lab (Mammen et al., 2002). The data set consisted of 200 frames collected with 1° oscillation range, 5.2 s exposure time, a wavelength of 1.04 Å and a crystal-to-detector distance of 100 mm.All three structures were refined with the PHENIX package (Adams et al., 2011) and model building was performed with Coot (Emsley et al., 2010). The quality of the three structures was checked with MolProbity (Chen et al., 2010), giving the following core/allowed statistics for the Ramachandran plot: 95.3/4.7% for the full-length structure, 97.6/2.4% for the P60_2LysM–chitohexaose structure and 97.4/2.6% for the P60_2LysM structure. [...] SAXS data were obtained at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. The data were recorded on beamline BM-29 and absolute-scale calibration was performed with bovine serum albumin (BSA) and water as references. The obtained data were azimuthally averaged, normalized and background-subtracted using the BsxCuBE software suite available at the beamline (Pernot et al., 2010). This yielded the scattering intensity I(q), where the scattering vector q is defined by q = 4πsin(θ)/λ, where θ is half of the scattering angle and λ is the wavelength of the incoming beam. All modelling was performed with CORAL (Petoukhov et al., 2012) and the scattering from all structures was evaluated with CRYSOL (Svergun et al., 1995). CORAL runs were made without any imposed symmetry and CRYSOL was run using the default settings. Both software packages are from the ATSAS suite v.2.4 (Petoukhov et al., 2012). […]

Pipeline specifications

Software tools PHENIX, Coot, MolProbity, CRYSOL, ATSAS
Applications Small-angle scattering, Protein structure analysis
Organisms Thermus thermophilus, Bacteria
Diseases Bacterial Infections
Chemicals Carbohydrates