Computational protocol: Structural and FunctionalAnalysis of the NLRP4 PyrinDomain

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[…] Native and SeMet crystals were grown at 20 °C using vapor diffusion in sitting drops containing equal volumes of protein solution (1.0 μL of the purified NLRP4 PYD at 1.5 mg/mL in concentrated size exclusion buffer I) and precipitant solution (0.1 μL of 0.2 M disodium tartrate and 2.2 M ammonium sulfate). Crystals formed after 4 days and were harvested, cryoprotected in paraffin oil, flash-frozen, and stored in liquid nitrogen. Diffraction data were collected at beamline BL14.1 at electron storage ring BESSY II (Berlin-Adlershof, Germany) operated by the Helmholtz-Zentrum Berlin (HZB) and processed using XDS. One SeMet and one native crystal were used to determine the structure. [...] Data were phased in SHELX, using 10 sites to 3.5 Å from the SeMet data set, which yielded an interpretable electron density map. The model was refined against the 2.3 Å native data set. Iterative cycles of refinement and model building were conducted in Coot and Phenix. The final model was refined to R and Rfree values of 0.207 and 0.246, respectively, at 2.28 Å resolution (Rfree was calculated using 5% of the reflections randomly omitted from the refinement). Data collection, model, and refinement statistics are summarized in Table . [...] The sequence alignment of the NLRP4 PYD with the PYDs of NLRP1, -3, -7, and -12, ASC2, ASC was generated in Multalin, and secondary structure comparison was completed using the on-line servers Dali and TopMatch-web.− Protein–protein interactions between the two monomers making up the asymmetric unit were evaluated using ProtorP. Structure visualization and analysis were performed in Pymol. Electrostatic surface potentials were calculated using APBS as part of the PIPSA (Protein Interaction Property Similarity Analysis) server to generate quantitative comparisons between PYD electrostatic surface potentials. […]

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