Computational protocol: Ribosome-dependent Vibrio cholerae mRNAse HigB2 is regulated by a β-strand sliding mechanism

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Protocol publication

[…] Crystallization and data collection for VcHigBA2, VcHigA2 and VcHigB2 have been described (). Complexes of VcHigB2 with nanobodies Nb2, Nb6 and Nb8 were prepared by mixing 1 mg of toxin with 1.3 mg of nanobody and isolating the pure complex by size-exclusion chromatography using the BioRad Enrich 70-10-30 column. Isolated complexes were crystallized using the hanging drop method. Drops consisting of 1 μl of protein solution (10 mg ml−1 in 200 mM NaCl, 20 mM TRIS–HCl pH 8.0) and 1 μl of precipitant solution were equilibrated against 110 μl precipitant solution. Various commercial screens were used: Crystal Screen and Crystal Screen 2 (Hampton Research), Morpheus, PACT premier and ProPlex (Molecular Dimensions) and Jena Classic (Jena Bioscience). All final crystallization conditions are listed in Table S1.All data were measured at the SOLEIL synchrotron (Gif-sur-Yvette, France) at 100K on beamline PROXIMA1 using a PILATUS 6M detector. All data were indexed, integrated and scaled with XDS (). The structure of the VcHigBA2 complex was determined using SAD phasing. Se-Met sites were located using SHELXD using the selenium anomalous signal () and phases were calculated using PHASER-EP (). Following density improvement with PARROT (), an initial model was automatically build with BUCCANEER (). The latter was used as a starting point for iterative refinement with phenix.refine () and manual rebuilding using Coot ().All other structures were solved by molecular replacement using PHASER-MR () and using the C-terminal domain of the VcHigA2 antitoxin or the VcHigB2 toxin as present in the VcHigBA2 complex as search models. To locate nanobodies, the co-ordinates of a nanobody against β-lactamase BcII and stripped of its complementarity-determining region (CDR) loops (), (PDB ID: 3DWT) was used as search model. In all cases, the structures were manually rebuilt using Coot and refined using phenix.refine. The final refinement cycles included TLS refinement (one TLS group per chain). Data collection and refinement statistics are given in . [...] SAXS data were collected at 15°C in HPLC mode at the SWING beamline at the SOLEIL synchrotron (Gif-sur-Yvette, France). The VcHigA2 sample (8 mg/ml in 20 mM TRIS–HCl pH 8.0 and 200 mM NaCl) was injected into a Shodex KW 402.5-4F column and ran at 0.2 ml/min. The data frames (1 ms exposure) were interrupted by a dead time of 0.5 ms. The buffer scatter was measured at the beginning of the chromatogram (during the dead volume), while the sample scatter was collected during the peak elution, which enables the acquisition of data at different protein concentrations. The data were processed with Foxtrot () and the ATSAS package (). After buffer subtraction, Guinier analysis was performed on each curve using the program AUTORG (). The curves of sufficient quality and stable Rg value along the elution peak were merged into a single final scatter curve used in further analysis. The molecular weights of the proteins and the protein complexes was estimated by using the QR ratio derived from the invariant volume of correlation ().A model of the VcHigA2 conformational ensemble was generated using the Ensemble Optimization Method (EOM) (). An initial pool of 10 000 random conformations was generated for the disordered N-terminal segment (residues 2–37) attached to the folded C-terminal domain based on the following assumptions: (i) the Cα distribution of the disordered segment is one found in random-coils, (ii) the symmetry of the folded core dimer is C2 (iii) no overall structure symmetry is imposed. A final ensemble was selected from the pool of conformations with the EOM algorithm based on the experimental scattering curve and this process was repeated five times. These final ensembles had χ2 values of 5.9, 1.1, 1.2, 1.2, 1.2 and consisted of 5–7 models. Based on the RG and DMAX values of the individual models all ensembles (except the first one) are structurally similar. The model ensemble with the lowest χ2 value (1.1) is considered to best represent the true ensemble and structural parameters of each model in this model ensemble are given in . The quality of the model ensemble was assessed using the RSAS and χ2FREE parameters as proposed recently () and is reported in . […]

Pipeline specifications

Software tools XDS, SHELX, Buccaneer, PHENIX, Coot, ATSAS, EOM
Applications Small-angle scattering, Protein structure analysis
Organisms Vibrio cholerae, Escherichia coli