To confirm the preferred formation of discrete Biot2 ─ FePP • Sav AA assemblies, we subjected crystals of Biot2 ─ FePP • Sav AA to X-ray crystallography. For this purpose, anaerobically grown Sav AA crystals were soaked with Biot2 ─ FePP for 48 hours. X-ray diffraction data of the dark red crystals (Figure ) were collected to a resolution of 1.74 Å (PDB: 7ZOF). The presence of iron in the crystals was confirmed by an X-ray fluorescence measurement at the Fe K-edge energy (~7112 eV) for iron (Figure ) before the collection of X-ray diffraction data. After the structure was determined by molecular replacement, inspection of the electron density maps revealed residual density in the biotin binding vestibule. Biot2 ─ FePP was modeled into the density with 80% iron occupancy (Figure ).