Protein purification

When possible, all purification steps were performed at 4 °C.

Cell pellets were thawed and resuspended in 10 % glycerol, 100 mM NaCl, and 20 mM Tris pH 7.5, then incubated for 1 h with 1 mg ml⁻¹ lysozyme. PMSF (1 mM) was added and cell suspensions were lysed by three passes through a microfluidizer. Intact cells were removed by low-speed centrifugation for 25 min at 12,500 rpm, in a JA-20 rotor (Beckman). The supernatant was centrifuged for 1 h at 200,000g in a Ti45 rotor (Beckman) and the insoluble material was discarded. The supernatant was spiked with 20 mM imidazole and incubated with Ni-NTA resin for 1 h with agitation. The resin was washed with (1) 1 M NaCl, PBS (pH 7.4) and 40 mM imidazole and (2) PBS and 60 mM imidazole. Protein was eluted after a 30-min incubation in PBS containing 320 mM imidazole, and was concentrated using a 10-kDa filter (Amicon) to 1 ml. Then, in the case of CBM70, the sample was run over an S200 16/60 gel filtration column equilibrated in PBS and the peak fractions were collected and used for αHA column preparation, or aliquoted and flash-frozen for storage; or, in the case of SNAP–CBM70, dialysed against PBS overnight, aliquoted, flash-frozen in liquid nitrogen and stored at −80 °C.

Cell pellets were processed as described above. After low-speed centrifugation, membranes were isolated from the lysate by centrifugation for 2 h at 200,000g in a Ti45 rotor, then collected, flash-frozen in liquid nitrogen and stored at −80 °C.

Membranes were thawed and resuspended in 300 mM NaCl, 20 mM Tris pH 7.5, 10 % glycerol, 40 mM imidazole, 1 % n-dodecyl-β-maltoside (DDM) and 0.1 % cholesterol hemisuccinate and incubated for 1 h with agitation. Aggregated material was removed by centrifugation at 200,000g for 30 min, and the supernatant was incubated with Ni-NTA resin for 1 h. The resin was washed with (1) 1.5 M NaCl, 20 mM Tris pH 7.5, 10 % glycerol, 40 mM imidazole and 0.1% LMNG and (2) 300 mM NaCl, 20 mM Tris pH 7.5, 10% glycerol, 80 mM imidazole and 0.1% LMNG. Protein was eluted after a 30-min incubation in 300 mM NaCl, 20 mM Tris pH 7.5, 5% glycerol, 400 mM imidazole and 0.05% LMNG, and was concentrated to 500 µl using a 100-kDa filter (Amicon), followed by overnight incubation on ice. The next day, the sample was run over a S6-increase 10/300 gel filtration column equilibrated in 100 mM NaCl, 50 mM Tris pH 7.5 and 0.025% LMNG. This buffer was supplemented with 5 mM MgCl₂ for KpsMT(E151Q)-KpsE preparations. The peak fractions were collected and concentrated to 2–3 mg ml⁻¹ using a 100-kDa filter, and were used for grid preparation or in vitro ATPase activity assays.

KpsMT-KpsE in complex with ADP–AlF₄⁻ was purified similarly. The concentrated Ni-NTA elution sample was dialysed overnight against buffer containing 100 mM NaCl, 50 mM Tris pH 7.5, 0.05% LMNG, 5% glycerol, 10 mM NaF, 2 mM AlCl₃, 5 mM ADP and 5 mM MgCl₂. The same buffer containing 0.025% LMNG and lacking glycerol was used to equilibrate the S6-increase gel filtration column. Peak fractions were collected on the basis of elution times, concentrated to around 2–3 mg ml⁻¹ and used for cryo grid preparation.