Computational protocol: A structural basis for Staphylococcal complement subversion: X-ray structure of the complement-binding domain of Staphylococcus aureus protein Sbi in complex with ligand C3d

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Protocol publication

[…] Sbi-IV–C3d complex (in 50 mM Tris pH 7.0 at ∼15 mg/ml) was subjected to a ProPlex screen and a tacsimate screen, using the ‘sitting drop’ vapour diffusion method. The Sbi-IV–C3d complex produced small needle-like crystals in various conditions of the ProPlex screen within 7 days. Crystals grew in the following ProPlex-96 conditions: 100 mM Tris pH 8.0, 20% (w/v) PEG 4000; 200 mM sodium chloride, 100 mM Tris pH 8.0, 20% (w/v) PEG 4000 and in 100 mM sodium HEPES pH 7.0, 1 M sodium citrate. Large crystals suitable for X-ray diffraction analysis were obtained in 100 mM Tris pH 8.0, 200 mM NaCl, 20% PEG 4000, using micro seading (Sead Bead, Hampton Research).X-ray diffraction data were collected at the Diamond Light Source (Oxfordshire, UK) on an ADSC Q315 3 × 3 CCD detector on station I04 (at wavelength λ = 0.9702 Å). A crystal of the Sbi-IV–C3d complex was removed from the crystallization drop using a cryoloop and was placed into cryoprotectant (20% (v/v) glycerol) containing reservoir solution for 1 min. The crystal was then removed from the drop using a micromount and held in a stream of gaseous nitrogen to facilitate freezing of the crystal. 360 images were collected at an oscillation angle of 1°. Data were processed using the HKL2000 package ().Molecular replacement was carried out with Balbes (), using the structure of C3d (Protein Data Bank accession code C3D1) as a search model. Automated rebuilding and refinement was carried out by Arp/wArp (). Model building was done with Coot () followed by rounds of refinement using Refmac5, part of the ccp4i software ().The final coordinates of the Sbi-IV–C3d complex have been deposited to the Protein Data Bank (PDB accession code 2wy8). [...] Synchrotron radiation X-ray scattering data were collected at the X33 beam line of the EMBL, Hamburg Outstation (DORIS III storage ring at DESY). Solutions of Sbi-E, Sbi-III–IV, Sbi-IV, C3d and complexes of the Sbi protein constructs with C3d were measured at protein concentrations of ∼2, ∼5, and ∼10 mg/ml (sample temperature 10 °C), using a MAR345 image plate detector and sample detector distance of 2.7 m and wavelength λ = 1.56 Å, covering the momentum transfer range 0.08 < s < 0.45 nm−1 (s = 4π sin(θ)/λ where 2θ is the scattering angle). Complexes of Sbi constructs were prepared in a 1:1 ratio at concentrations mentioned above. Prior to data collection, dynamic light scattering analysis (Nano-S Zetasizer, Malvern) was used to ensure the monodispersity of the protein samples. To check for radiation damage, two successive 2 min exposures taken on the same sample were compared; no radiation effects were observed. The data were processed using standard procedures and extrapolated to zero solute concentration using the program package PRIMUS ().The forward scattering I(0) and the radius of gyration Rg were computed from the entire scattering patterns using the indirect transform package GNOM (), which also provided the intraparticle distance distribution function p(r) and the maximum dimension Dmax. The molecular mass of the solute was evaluated by comparison of the forward scattering with that from a reference solution of bovine serum albumin (molecular mass 66 kDa). The estimation of excluded volume Vex and low resolution ab initio models of Sbi-E, Sbi-III–IV, Sbi-IV, C3d and complexes thereof were obtained using the program DAMMIF (). The program employs simulated annealing to build a compact interconnected configuration of beads inside a sphere with the diameter Dmax that fits experimental data minimizing the discrepancy:χ2=1N−1∑jIexp(sj)−cIcalc(sj)σ(sj)2where N is the number of experimental points, c is a scaling factor, Iexp(sj) and Icalc(sj) are experimental and calculated intensities, respectively, and σ(sj) is the experimental error at the momentum transfer sj. Ten DAMMIF runs were performed to check the stability of the solution, and the results were averaged using the program DAMAVER () to yield the most probable models. Rigid body modeling was performed using the program SAXREF (). […]

Pipeline specifications

Software tools ATSAS, DAMMIF
Application Small-angle scattering
Organisms Dipturus trachyderma, Staphylococcus aureus, Epipremnum aureum