Computational protocol: Structures of the NLRP14 pyrin domain reveal a conformational switch mechanism regulating its molecular interactions

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[…] SeMet-labelled NLRP14 PYD and native NLRP14 PYD mutants (D86V and L84R) were crystallized at 20°C in sitting drops using vapour diffusion. SeMet-labelled crystals were obtained by mixing equal volumes of protein solution (1.0 µl purified NLRP14 PYD at a concentration of 8.5 mg ml−1 in gel-filtration buffer) and precipitant solution (1.0 µl 0.1 M HEPES pH 7.5, 2.6 M ammonium sulfate, 2% PEG 400). NLRP14 PYD D86V was crystallized with a precipitant solution consisting of 0.2 M cesium chloride, 2.2 M ammonium sulfate and NLRP14 PYD L84R was crystallized with 0.2 M ammonium acetate, 2.2 M ammonium sulfate at protein concentrations of 5.7 and 8.5 mg ml−1, respectively. Crystals formed after 7 d and were prepared for cryo-crystallography by a quick bath in either LV CryoOil (MiTeGen) or 3.4 M sodium malonate pH 7.0. Flash-cooled crystals were stored in liquid nitrogen. Diffraction data were collected on beamline BL14.1 at the BESSY II electron-storage ring, Helmholtz Zentrum Berlin für Materialien und Energie and on the microfocus beamline ID23-2 at ESRF Grenoble (Gabadinho et al., 2010). Data were processed using iMosflm v.7.0.6 (Battye et al., 2011). [...] The NLRP14 PYD structure was solved using the 4W-MAD protocol of Auto-Rickshaw (Panjikar et al., 2005). The input diffraction data were prepared and converted for use in Auto-Rickshaw using programs from the CCP4 suite (Collaborative Computational Project, Number 4, 1994; Winn et al., 2011). F A values were calculated using SHELXC (Sheldrick, 2008, 2010). Based on an initial analysis of the data, the maximum resolution for substructure determination and initial phase calculation was restricted to 3.2 Å. 13 out of 16 Se sites were found using SHELXD (Schneider & Sheldrick, 2002). The correct hand for the substructure was determined using ABS (Hao, 2004) and SHELXE (Sheldrick, 2010). The occupancy of all substructure atoms was refined and the initial phases were calculated using MLPHARE (Collaborative Computational Project, Number 4, 1994). The twofold noncrystallographic symmetry (NCS) operator was found using RESOLVE (Adams et al., 2010). Density modification, phase extension and NCS averaging were performed using DM (Cowtan, 1994). A partial α-helical model was produced using HELICAP (Nam et al., 2004). NLRP4 PYD (PDB entry 4ewi; Eibl et al., 2012) was used as a template for further model building. The partial model contained 385 residues out of the total of 424 residues. Iterative cycles of model building and refinement using Coot (Emsley & Cowtan, 2004) and PHENIX (Adams et al., 2010) led to a final model with an R cryst and R free of 0.205 and 0.261, respectively, at 2.4 Å resolution (R free was calculated using 5% of the reflections, which were randomly omitted from the refinement).The D86V and L84R mutant structures of NLRP14 PYD were both solved by molecular replacement using Phaser from the CCP4 suite (McCoy et al., 2007; Collaborative Computational Project, Number 4, 1994; Winn et al., 2011). Wild-type NLRP14 PYD from Ser6 to Pro67 corresponding to α-helices 1–4 served as a model. Thus, calculation of an OMIT map rules out that the interesting α-helix 5–6 region is biased by the atomic model. Finally, iterative cycles of model building and refinement using Coot (Emsley & Cowtan, 2004) and PHENIX (Adams et al., 2010) led to a model of NLRP14 PYD D86V with an R cryst and R free of 0.216 and 0.268, respectively, at 3.0 Å resolution. The final model of NLRP14 PYD L84R at 2.0 Å resolution was refined to an R cryst and R free of 0.182 and 0.222, respectively (R free was calculated using 5% of the reflections which were randomly omitted from the refinement). Table 1 summarizes the data-collection, model and refinement statistics of wild-type and mutant NLRP14 PYD. The final model of NLRP14 PYD includes four molecules (chain A, Ser7–Ile95; chain B, Ser8–Gln100; chain C, Ser7–Asn96; chain D, Ser6–Ala99). […]

Pipeline specifications

Software tools iMosflm, CCP4, SHELX, Coot, PHENIX
Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens