On-Column Glutaraldehyde Cross-Linking of UtpB

The complex was on-column glutaraldehyde cross-linked28 (link) in 50 mM HEPES-NaOH pH 7.7 (4 °C), 200 mM NaCl, 1 mM EDTA as follows: 500 μl of a 0.25% glutaraldehyde solution was pre-injected onto a size-exclusion column (Superose 6 Increase 10/300 GL, GE Healthcare) and run for 20 min at a flow rate of 0.25 ml/min. The run was paused, the injection loop vigorously washed, UtpB injected, and the run was continued at the same flow rate. The on-column cross-linking procedure was optimized with UtpB purified using affinity tags on different subunits (YSK43 and YSK47, Supplementary Data 1). UtpB from both strains exposed to the pre-injected glutaraldehyde bolus exhibited the same elution behaviour as the non cross-linked complex (Supplementary Fig. 5a) and the protein complex eluted at 13.14 ml.