Extended Data Fig. 2: SdeA(231–1190) is an active monomer in solution.

a, SdeA, wild-type SdeA(231–1190), SdeA(231–1190)^E860A/E862A or SdeA(231–1190)^H277A were incubated with NAD⁺ and Ub in the presence or absence of His–RAB33B. Ubiquitinated His–RAB33B were analysed using tricine gels, Coomassie staining and immunoblotting with anti-His and anti-Ub antibodies. b, SdeA(231–1190) and RAB33B(15–202) were incubated with GST–Ub and NAD⁺, and self-ubiquitinated SdeA was detected by Coomassie staining, immunoblotting with anti-Ub antibodies, and Pro-Q diamond phosphoprotein staining. c, SdeA(231–1190), NAD⁺ and RAB33B were incubated with 0, 1, 2 or 5 mM NADH. The ubiquitination reactions were analysed using tricine gel and Coomassie staining. d, Analytical ultracentrifugation results showed that SdeA(231–1190) is a monomer. Analytical ultracentrifugation analysis yielded a sedimentation coefficient of 5.13 S, and a molecular mass of approximately 106 kDa. The buffer is 10 mM Tris pH 8.0, 200 mM NaCl and 5 mM DTT. e, Gel filtration profile of the SdeA(231–1190) protein and the molecular markers on Superdex-75 column (GE Healthcare) are shown. The sizes of the molecular markers are marked on top of the peaks. The samples of SdeA(231–1190) collected from the Superdex-75 column were run on SDS–PAGE gels and detected by Coomassie staining. a–e, Similar results were obtained in three independent experiments. a–c, e, Uncropped blots and gel images are shown in Supplementary Fig. 1. f, Two views of the superimposition of the structures of the two molecules in the asymmetric unit, coloured in different colours. g, Structure of the CTD region in the crystallized protein can be divided into two parts (left and right). The α helices are numbered according to their orders in the residue region from 908 to 1190. h, Topological diagram of the CTD region shown in g. The N and C termini of the pCTD domain are labelled.