a, Position of the bound SO 4 ion as in the NEK7 crystal structure25, superimposed here in the structure of the NLRP3–NEK7 complex. b, Cryo-EM densities (5σ) at the NLRP3–NEK7 interfaces. Views correspond to those of Fig. 3b, c. c, Amylose pulldown of wild-type and mutant NEK7 without NLRP3, showing input and precipitate gels, which serve as negative controls for Fig. 3d. Experiments were repeated 3–6 times. d, Input gel for amylose pulldown of wild-type and mutant NEK7 shown in Fig. 3d. Experiments were repeated 3–6 times. e, Alternative calculation of the percentage of NEK7 pulldown relative to wild type, by subtracting the NEK7/NLRP3 ratio in the absence of NEK7 from the observed NEK7/NLRP3 ratio (shown as mean ± s.d. for n = 3–6 experiments). f, Cytoplasmic LDH released into the supernatant, quantified by comparison with total intracellular LDH of untreated cells (triton-X-lysed). Data are presented as mean ± s.d. for n = 3 replicates from 2 independent experiments. g, Top, mapping the NEK9-binding site onto the structure of the NEK7–NEK9 complex (PDB code 5DE2)29. NEK7 and NEK9 are shown in yellow and cyan, respectively, with the NEK9-binding residues of NEK7 highlighted in red. Bottom, a rainbow-coloured NEK7 structure showing that the NEK9-binding residues are from the first part of the NEK7 C-lobe. h, Mapping the NLRP3-binding site (red) onto NEK7 in the NLRP3–NEK7 structure. The NLRP3-binding site overlaps with the NEK9-binding site on NEK7. i, Superposition of NEK7 back-to-back dimer (yellow and pink) in the NEK7–NEK9 complex structure (PDB code 5DE2)29 onto the NEK7 monomer in the NLRP3–NEK7 structure. The pink monomer in the NEK7 dimer clashes with NLRP3, which suggests that NEK7 dimerization cannot occur in the NLRP3–NEK7 complex. j, NLRP3-knockout iBMDMs were reconstituted with wild-type or mutant human mNG-tagged NLRP3, primed by LPS (4 h) and stimulated by nigericin (30 min). LDH release was analysed as in f. Dots, individual data points. Data are presented as mean ± s.d. for n = 3 replicates from 2 independent experiments. k, LRR phosphorylation at Y85930, which might cause steric and charge repulsion with NEK7. l, NLRP3 activation model and NEK7 interactions. The hypothetical ~90° rotation of the NBD and HD1 of NLRP3 upon activation moves the NBD away from the NEK7 interaction region (indicated by a box). Source data