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. 2021 Jan 8;12:188. doi: 10.1038/s41467-020-20319-5

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Domain organizations of human NLRP1, CARD8. Death domains (PYD and CARD) are highlighted in blue or red. b Percentage of ASC-GFP specks induced by different amounts of mCherry-tagged NLRP1-FIIND^UPA-CARD (red) or mCherry-tagged NLRP1-CARD (light red) in HEK293T-ASC-GFP cells. Cells were fixed 24 hours after transfection for fluorescence microscopy. Data are presented as mean values ± SD. P value was calculated with one-way ANOVA, n = 3 biological replicates. ‘*/**/****’ indicates P value < 0.05, 0.01, and 0.0001, correspondingly. c Corresponding western blot of mCherry-tagged NLRP1 constructs in Fig. 1b. d Blue-native PAGE blot of mCherry-tagged NLRP1-FIIND^UPA-CARD and mCherry-tagged NLRP1-CARD. Yellow arrows, monomeric species. e Representative negative stain EM images recombinant NLRP1-FIIND^UPA-CARD and NLRP1-CARD at various concentrations. f A summary of oligomer formation of various NLRP1 constructs at various concentrations. g Two of the best 2D class images with clearly distinguishable features of recombinant NLRP1-FIIND^UPA-CARD filamentous complex, from negative stain EM analysis. h A side view and a top view of the 3D density model at estimated 20–30 Å resolution (after 3D refinement using RELION) generated based on particles extracted from the 15 nm 2D class. A similar heterogenous helical symmetry can be found in some corn cobs. i Negative EM images of SUMO-tagged NLRP1-FIIND^UPA. The ring-complexes are –20 nm in size. j Proposed model for NLRP1 FIIND^UPA-CARD oligomers.