Extended Data Fig. 6: Preparation of GSDMB pore samples and cryo-EM data processing.
From: Structural mechanisms for regulation of GSDMB pore-forming activity
a–d, Reconstitution, purification, and EM examination of GSDMBIso4 pores. a, Schematic diagram illustrating reconstitution and extraction of GSDMB pores from the liposomes. b, Purification of GSDMB pores by gel-filtration chromatography. The highlighted fraction containing homogenous pores were analyzed by SDS-PAGE and also applied to negative-stain EM examination and final cryo-EM data collection. Negative-stain EM image of GSDMB pores (representative of three independent experiments) is shown. Scale bar, 100 nm. c, Cryo-EM micrograph of GSDMB pores (representative of 7790 micrographs). Scale bar, 50 nm. d, Percentages of GSDMB pores with C26, C27, C28, C29 and C30-fold symmetry among a total of 270,000 top-view pore particles. e, Pore size distribution for GSDMBIso3. Inner diameters of a total of 8,600 GSDMBIso3 pores on the liposomes were measured. f, Processing of the cryo-EM dataset for GSDMB pore structure determination. Briefly, initial 3D model reconstruction generated a correct pore-like model. Iterative 2D and reference-guided 3D classifications were performed to select the best particles. A further round of 3D classification of the best particles indicates symmetry heterogeneity in the pores; a certain class of symmetry was chosen for final structural determination. Deep 2D classification allowed separation of the side view particles that were combined with best top view particles for 3D reconstruction and symmetry-imposed refinement to yield final cryo-EM maps.
