a, Analytical size-exclusion chromatography and SDS–PAGE analysis of crystallization samples of the humanized rat SUCNR1–Nanobody6–NF-56-EJ40 complex. Shown is a typical result from n = 2 independent experiments. Although partial complex aggregation was observed, this did not interfere with crystallization. For gel source data, see Supplementary Fig. 1b. b, Top, initial crystallization hits for the humanized rat SUCNR1–Nanobody6–NF-56-EJ40 complex, shown in normal (left) and cross-polarization (right) imaging modes. Bottom, optimized crystals used for data collection shown in normal (left) or cross-polarization (right) imaging modes. A typical result from n = 3 independent experiments is shown. c, The 2F o − F c electron density map contoured at 1.5σ for a part of Nanobody6. d, The 2F o − F c electron density map contoured at 1.5σ for helix VII in humanized rat SUCNR1. e, F o − F c composite omit map (top) for NF-56-EJ40 and glycerol contoured at 1.5σ is shown in orange. The 2F o − F c map (bottom) for NF-56-EJ40 and glycerol after refinement contoured at 1.5σ is shown in blue. f, Top view of apo rat SUCNR1 (shown in orange) and humanized rat SUCNR1 (shown in blue) in complex with NF-56-EJ40 (shown as green sticks). Large structural rearrangements are indicated by red arrows. Note that ECL2 is completely structured in the humanized rat SUCNR1 structure. g, Top view of the NF-56-EJ40-binding site in humanized rat SUCNR1 overlaid with apo rat SUCNR1. Important side chains around NF-56-EJ40 (shown in green) are shown as sticks and are coloured blue for humanized rat SUCNR1 or orange for apo wild-type rat SUCNR1. For clarity, only the backbone of the humanized rat SUCNR1 is shown in cartoon representation. h, Side chains that directly interact with NF-56-EJ40 via hydrogen bonding, π–π stacking and cation–π stacking are listed in black. The hydrogen-bonding interactions are shown by black dashed lines and the π–π and cation–π interactions are shown by green dashed lines. Additional residues with van der Waals interactions are listed in green, and their interaction surfaces are indicated by solid green lines. i, Top view of the humanized rat SUCNR1 (blue) in complex with NF-56-EJ40 (green sticks) and of the apo rat SUCNR1-derived model of human SUCNR1 (red) with the binding mode of NF-56-EJ40 (pink) from molecular-docking studies, for which details are shown in Extended Data Fig. 7a. Note how both NF-56-EJ40 poses differ considerably, as indicated by red arrows. j, Detailed views of the NF-56-EJ40-binding site. For clarity, only side chains are shown. Humanized rat SUCNR1 is shown in blue; wild-type rat SUCNR1 is shown in orange. Note the side-chain flips for R953.29, L983.32, H993.33 and Y171 between both structures. k, Top view of wild-type rat SUCNR1 structure (left), the apo rat SUCNR1-based homology model of human SUCNR1 in complex with NF-56-EJ40 (middle) and the humanized rat SUCNR1 structure in complex with NF-56-EJ40 (right). The surface is shown coloured by electrostatic charge. The two key positions (K/E1.31 and K/N7.32) are highlighted by arrows. NF-56-EJ40 is shown in yellow as a ball-and-stick model. Note the differences between the NF-56-EJ40-binding mode determined in the modelled structure and in the crystal structure, and between the surface charge distributions in rat, human and humanized rat SUCNR1. Source Data