SyrB2 and its mutants were buffer exchanged into RXN buffer three times using Amicon™ Ultra 30 kDa MWCO centrifugal filter units (Millipore Sigma) after initial immobilized metal affinity chromatography. Concentrated protein was diluted to ~8 mL with RXN buffer and filtered with 0.22-micron filters prior to loading into a superloop connected to an AKTA Pure protein purification system (Cytiva) for size-exclusion chromatography. A HiLoad 26/600 Superdex 200 pg column (Cytiva) was pre-equilibrated with RXN buffer. Protein was then applied and eluted (flow = 1 mL/min) over three injection/elution cycles. Relevant fractions, as determined by SDS-PAGE, were pooled and concentrated in the Amicon™ Ultra 30 kDa MWCO centrifugal filter units. Protein was aliquoted, flash frozen in liquid nitrogen, and stored at -80°C until further use.