Figure 1: Unliganded and complexed structures of ZNRF3 and Rspo proteins.

(a) Schematic domain organization of Rspo (top) and ZNRF3/RNF43 proteins (bottom) roughly at scale. The domains included in the crystallization constructs are coloured in blue, red and orange. Disulphides are derived from the crystal structure, except for those of the TSR domain of Rspo, which are based on a model⁴⁸. (b) Cartoon representation of the fold of the ZNRF3 ectodomain protomer. β-strands are numbered and α-helices are labelled in alphabetical order from the N to C terminus. (c) Structure of the recurring ZNRF3_ecto dimer with view parallel to the putative membrane layer and from top towards the membrane. An acidic region with sequence ¹⁰⁵NNNDEEDLYEY¹¹⁵ is highlighted in red in b and c. (d) The xRspo2_Fu1–Fu2 structure. Both β-hairpins and disulphide bridges line up to form a ladder-like structure. The second β-hairpin of Fu1 contains an exposed methionine side chain. (e) Fu1 and Fu2 share the same architecture, except that the second β-hairpin of Fu1 is considerably longer. (f) The ZNRF3_ecto–Rspo2_Fu1–Fu2 complex as the same 2:2 symmetric complex in all seven crystallographic observations. Shown are two views parallel to the putative membrane orientation. The RNF43_ecto–Rspo2_Fu1–Fu2 complex resembles one half of this complex (Supplementary Fig. S5). (g) The ZNRF3_ecto–Rspo2_Fu1–Fu2 interface. xZNRF3_ecto is shown in semi-transparent surface (orange) and ribbon, xRspo2_Fu1–Fu2, is depicted in blue. Residue side chains involved in the interface are shown as sticks and labelled (atom colouring: dark blue, nitrogen; red, oxygen; yellow, sulphur). Dotted lines represent hydrogen bonds. A corresponding stereo figure with final electron density can be found in Supplementary Fig. S6. (h) The Met-finger pocket. Structural features are represented as in g. BR, basic region; PAD, protease-associated domain; SP, signal peptide; TM, transmembrane; TSR, thrombospondin-related domain.