Extended Data Fig. 1: Mouse genotyping, confocal imaging of spermatozoa and endogenous purification of the CatSpermasome from mouse sperm.

a, The genotype of each mouse was verified by two PCR reactions. Top, schematic of the genotyping procedure. Bottom, a group of representative PCR results. NC, negative control (empty template); WT, wild type; KI/+, heterozygote knock-in; KI/KI, homozygote knock-in. In total, 49 wild-type, 676 KI/+, and 207 KI/KI mice were verified. b, An drawing of a mouse sperm. CatSper was mainly distributed at the principal piece of spermatozoa. c, EGFP fluorescence was detected in the principal piece (red arrows) of knock-in mouse spermatozoa, but not wild-type mouse spermatozoa. Blue arrows, auto-fluorescence signal observed in the middle piece of spermatozoa. Shown here are one of the two images taken for each sample. Scale bars, 30 μm. d, Schematic of CatSpermasome purification. e, The purified protein sample was subjected to gel filtration analysis. The peak fractions of CatSpermasome (arrow) were collected and concentrated for cryo-EM and MS studies. Inset, the cross-linked protein sample was visualized on SDS–PAGE by silver staining. The corresponding protein band (arrow) in a separate gel without staining was cut out for MS analysis. For gel source data, see Supplementary Fig. 1. f, A representative EM micrograph of the CatSpermasome sample stained with uranyl acetate (one micrograph out of five in total for the negative staining sample). Scale bar, 50 nm.