Extended Data Fig. 1: Preparation of full length, wild-type human NLRP3 for electron microscopy analyses.
From: Structure of the NLRP3 decamer bound to the cytokine release inhibitor CRID3
a, Analytical SEC of recombinant, human MBP–NLRP3 (3–1036) reveals two elution peaks, one close to the void volume (peak 1) and one at a size larger than 1 MDa (peak 2). The peak ratio is shifted from peak 1 under standard buffer conditions (50 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1 mM ADP, 0.5 mM TCEP) towards peak 2 by addition of CRID3 (10 µM CRID3). Analytical SEC runs were performed on a Superose 6 Increase 10/300 GL column with 700 µl applied at a concentration of 3 mg/ml (repeated >3 times). b, Coomassie-stained SDS PAGE analysis of purified MBP-NLRP3 (repeated >3 times). c, SEC–MALS experiment of MBP-NLRP3 Peak 2 plus ADP on a Superose 6 Increase 10/300 GL column. The calculated mass of an MBP-NLRP3 monomer is 158 kDa. d, SEC–MALS experiment of MBP-NLRP3 Peak 2 plus ADP plus CRID3 on a Superose 6 Increase 10/300 GL column. The average apparent mass of Peak 2 from both experiments equals 10-times the calculated mass of the protein. e, Analytical SEC of recombinant, human NLRP3 (3–1036) after TEV digestion using a Superose 6 Increase 3.2/300 column. f, Negative-stain micrograph of TEV-digested, gel filtered NLRP3 plus CRID3 plus BS3 Peak 2 elution fraction that was used for subsequent cryo-EM grid preparation (right, one among a few hundred images). An SDS-PAGE analysis of the protein w/o BS3 is shown left. The scale-bar equals 200 nm. g, Exposure of NLRP3 peak 2 to ATP does not convert the decamer assembly into another state. MBP-NLRP3 expressed in Sf9 cells and purified by affinity chromatography in the absence of any nucleotide elutes in two peaks (top). Incubation of MBP-NLRP3 peak 2 (from top) with 1 mM ATP•Mg2+ for 2 h at 4 °C does not lead to a change in the elution profile, indicating the autoinhibition of the decamer assembly (bottom). h, THP-1 cells contain an oligomeric endogenous NLRP3 species. Immunoblotting of BN-PAGE of THP-1 cell lysate supernatant and pellet fractions from PMA-differentiated, untreated and LPS-treated wild-type cells pre-incubated with or without CRID3 (repeated 2 times). The supernatant of LPS-treated and BS3 cross-linked cell lysates contains an oligomeric NLRP3 fraction in the presence or absence of CRID3 (left). In the pellet fractions of LPS-treated cell lysates only an oligomeric NLRP3 species was detected at a molecular mass around 1.15 MDa, in line with the formation of a decamer (middle). Anti-NLRP3 Cryo-2 and anti-β actin antibodies were used for the WB analysis. BN-PAGE analysis of recombinant human (MBP-)NLRP3 protein expressed in Sf9 cells and purified by affinity chromatography and SEC (right). Source data are provided as a source data file.
