Methods of experientation

Cloning, expression and purification of the MCC complex

Schizosaccharomyces pombe CDC20–SLP1, MAD2 and MAD3 genes amplified by PCR using an S.pombe cDNA library pTN-TH7 as a template and a pFBDM vector.

A double Strep-tag II together with TEV cleavage site were introduced into N terminus of Cdc20-slp1 and Leu12Ala and Arg133Ala mutations where introduced into Mad2.

Proteins were recombined with the DH10MultiBac. The complex was expressed using the baculovirus and insect cell systems.

Crystallization, data collection and structure determination

Hanging drop method was utilized to obtain crystals. They did this by pre-incubating 1:1 v/v of 4.5 mg ml⁻¹ of protein with the crystallization solution, 100 mM Tris-HCl pH 8.8, 21% PEG 3350, 30% ethylene glycol, 5 mM dithiothreitol (DTT), and 5 mM EDTA at 20 °C for 24 h, followed by streak seeding. Crystals grew after 2 weeks and were mounted in 0.2-0.3 mm cryoloops and frozen in liquid nitrogen. Native crystals diffracted to a minimum Bragg spacing (d_min) of about 2.1 Å. The diffraction data set was collected at the I02 beamline of Diamond Light Source, it was processed with XDS and scaled to 2.3 Å with SCALA. Phase information was gathered by molecular replacement with AMoRe. Monomeric coordinates from the crystal structures of human MAD2(L13A) (PDB 2VFX), human BUBR1 (PDB 2WVI) and human WDR5 (PDB 3EMH) were used as search models

APC/C ubiquitination assays with wild-type and mutant Cdh1

Saccharomyces cerevisiae Cdh1 mutants were obtained using PCR-based mutagenesis and cloned into a linear pRSET vector. APC/C ubiquitination assays were adopted and modified S-labelled Clb2p and securin (Pds1p) and unlabelled Cdh1 mutants were prepared using TNT T7 Quick-coupled in vitrotranscription (or translation). Reactions were incubated at room temperature for 15 min and were analysed using 8% SDS–PAGE. Gels were fixed and stained with Coomassie blue, then dried and exposed to BioMax MR Film (Kodak).

Native gel electrophoresis

Correct folding of S. cerevisiae Cdh1 mutants was assessed by their co-migration with the APC/C in native gel electrophoresis. 50 ng of apoAPC/C was mixed with 2 μl of ³⁵S-labelled IVT-produced Cdh1 and 0.7 μl of 100 mM CaCl₂ in a volume of 14 μl with 10 mM Tris pH 8.0, 150 mM NaCl, 3 mM DTT, 1 mM magnesium acetate and 2 mM EGTA. The entire reaction was loaded onto a 5.25% non-denaturing polyacrylamide gel run at 4 °C, 110 V for 2 h. Gels were fixed and stained with Coomassie blue, then dried and exposed to film.

KEN–D and D-KEN peptides

The KEN–D, D–KEN, KEN–D^mut  and KEN^mut–D peptides were designed with a 17-residue linker between the KEN-box sequence (NKENEGPA) and the D-box sequence (QRAALSDITNS), or between their respective mutant sequences (NSASEGPA and QSAAASDITSS). The peptides were synthesized by Cambridge Peptides.

Fitting structure coordinates into the human APC/C^MCC EM map

The structure coordinates of S. pombe Cdc16–Cdc26 complex (PDB 2XPI), Cdc27 (PDB 3KAE), Apc10 (PDB 1GQP), modelled Apc2 and MCC were fitted into the human APC/C^MCC map (EMD-1591) based on the S. cerevisiae APC/C assignment, using UCSF Chimera.