Extended Data Fig. 1 Biochemical characterization of the phosphate-binding pocket of cyclin B1. a, Size exclusion chromatography (SEC) runs on untagged cyclin B1158-433 wild-type (black) or pocket mutant (red) resulted in nearly identical elution volumes of approximately 1.83 ml, indicating that these complexes run as monomeric protomers with an identical fold. b, Far UV circular dichroism (CD) spectra of cyclin B1158-433 wildtype (black) or pocket mutant (red) at roughly 5 μM concentration. c, Thermal-shift assays performed with cyclin B1158-433 wild-type (black) or pocket mutant (red). The apparent melting temperatures are nearly identical at 36°C. d, MBP pull-downs using recombinant CDK1 with an N-terminal MBP tag and C-terminal twin-strepII tag, cyclin B1wt/mut with a C-terminal 8xHis tag and purified CKS1. Both complexes pull down CKS1 efficiently. MBP served as negative control. e, MBP pull-downs using recombinant MBP-tagged CDK1/cyclin B1wt/mut complexes with the same tags as described in panel (d) and purified securinΔ160-separaseC2029S. Only CDK1/cyclin B1wt pulls down phosphorylated separase efficiently, whereas CDK1/cyclin B1mut fails to pull down separase. MBP served as negative control. f, SEC runs on the isolated securinΔ160-separaseC2029S (black dashed line), the isolated securinΔ160- separaseC2029S/S1126T (solid yellow line) and the CDK1/CKS1/cyclin B1 complex (solid blue line) resulted in elution volumes of approximately 1.46 ml, 1.46 ml and 1.6 ml, respectively. Adding ATP and the CDK1/CKS1/cyclin B1wt complex to securinΔ160- separaseC2029S/S1126T results in a stable interaction between these two complexes (solid red line), due to phosphorylation of T1126 by CDK1. This is indicated by a clear shift towards higher molecular weight with an elution volume of roughly 1.4 ml. However, adding ATP is dispensable for complex formation between separase and the CCC complex in vitro when using a securinΔ160-separaseC2029S/S1126E mutant version (solid green line). Of note, in CCC complex CDK1 has a C-terminal 8´His tag, cyclin B1 a Cterminal twin-strepII tag and CKS1 no tag.