a, Cdc50p from E2Pinhib, shown in rainbow colours. C α carbons of glycosylated asparagine residues are shown as black spheres; Drs2p is shown in grey. b, Position of the disulfide sites in Cdc50p from E2Pinhib. c–e, Glycosylation sites in Cdc50p from E2Pinhib. Map levels are 1.75 r.m.s.d. in c and d. The density for the glycosylation at Asn237 is only apparent at lower map levels (0.75 r.m.s.d.) and has not been modelled. f, Alignment of Cdc50p (magenta cartoon) and a monomer of human seipin57, a lipid-binding protein (blue cartoon, PDB 6DS5), illustrating the similar folds (although some loops of Cdc50p are more extensive). The sequence identity between the two proteins is only 4%. Transmembrane helices of seipin that extend from similar positions to the helices of Cdc50p are not present in the structure, but may extend into the membrane in a similar manner to that observed for Cdc50p. g, The transmembrane helices of the Drs2p–Cdc50p complex viewed from the luminal side. The transmembrane helices of Drs2p are rainbow coloured; Cdc50p transmembrane helices are pink. h, Interaction between the Cdc50p ectodomain (shown as surface) and the luminal TM3–TM4 loop of Drs2p (light green). i, Interaction between the N terminus of Cdc50p and the segment of Drs2p that leads from TM4 to the phosphorylation site. Residues 529–538 are not present in P2-ATPases and are shown in dark blue. j, Segments of Drs2p that were found to interact with Cdc50p mutants that disrupt the formation of the complex are highlighted in green, and the insert in Drs2p between TM4 and the phosphorylation site is blue. The structure shown in h–j is E2Pinhib. Cdc50p is coloured on the basis of the conservation using ConSurf58. k, Part of a sequence alignment of proteins of the CDC50 family from S. cerevisiae (Cdc50p, Lem3p and Crf1p) and human CDC50A and CDC50B. Residues that are important for complex formation are identified. Uniprot identifiers are as follows: Cdc50p, P25656; Lem3p, P42838; Crf1p, P53740; CDC50A, Q9NV96; CDC50B, Q3MIR4. The alignment was performed using Clustal Omega59. For full sequence alignment, see Supplementary Fig. 1.