2.1. Macromolecule production  

The PaFMO protein was purified from P. aestuarii cell cultures following established protocols (Orf et al., 2014 ▸). Briefly, the cells were disrupted by sonication, centrifuged at low speed to remove debris and centrifuged again at 186 500g for 2 h in a buffer consisting of 20 mM Tris pH 8.0. The membrane-containing pellet was resuspended in the same buffer after centrifugation, and sodium carbonate was added through dialysis to a final concentration of 0.4 M over at least 24 h. The solution was then centrifuged at 307 500g for 2 h, after which the supernatant was collected and extensively dialyzed into 20 mM Tris pH 8.0. The dialyzed material was loaded onto Q Sepharose resin (GE) and elution was carried out with an NaCl gradient, with PaFMO typically eluting at NaCl concentrations of >300 mM. Fractions containing PaFMO were pooled and concentrated before loading them onto a Superdex S75 gel-filtration column (GE) equilibrated with 20 mM Tris, 40 mM NaCl pH 8.0. The peak from the gel-filtration column was collected and concentrated before final purification on a HiTrap Q column (GE). The purified PaFMO protein was then concentrated to 12 mg ml⁻¹ for crystallo­graphic studies.