Fig. 1: The structure of FMA bound in the ligand-binding surface pocket of NanoLuc luciferase.

a 2Fo-Fc electron density (contour level 1.2 σ) at the FMA-oxyluciferin binding site. b Cartoon representation of the overall structure of NanoLuc asymmetric dimer (chain A in cyan and chain B in blue) with bound FMA luciferin (yellow). c Superposition of chain A (cyan) and chain B (blue). The structural element responsible for symmetry breaking, encompassing helix H4, loop L7 and strand S4, is colored orange. d B-factor putty representation of NanoLuc homodimer. e Dimer interface visualization. All residues involved in the dimer interface are shown as space-filling spheres. FMA is shown as yellow spheres. f Chord plot showing the interactions between chains A and B in the NanoLuc dimer at the secondary structure level, calculated and visualized by Protein Contact Atlas⁸³. g Cutaway surface representation of FMA-bound NanoLuc dimer. h Close-up view of FMA-binding pocket with residues creating the active site in stick representation. Key hydrogen bonds are shown as dashed yellow lines. i Sequence alignment between NanoLuc and the catalytic unit of O. gracilirostris luciferase (OLuc). Secondary structure elements found in NanoLuc are shown above the alignment. Amino acid residues mutated during the NanoLuc engineering⁸ are labeled with the red dot. The numbering indicated above the alignment corresponds to the NanoLuc structure (PDB ID: 5B0U)¹⁷.