Atpγs

Wild-type human LONP1 was diluted to a concentration of 2.5 mg/ml in 50 mM Tris pH 8, 75 mM KCl, 10 mM MgCl₂, 1 mM TCEP, and 1 mM ATPγS (Jena Bioscience, purity ≥90% (HPLC), contains <10% ADP). Samples were mixed and incubated on ice for 5 min to ensure nucleotide binding. Four microliters of the sample were applied onto 300 mesh R1.2/1.3 UltrAuFoil Holey Gold Films (Quantifoil) that were plasma cleaned prior to sample application for 7 s using a Solarus plasma cleaner (Gatan, Inc.) with a 75% nitrogen, 25% oxygen atmosphere at 15 W. Excess sample was blotted away for 4 s using Whatman No. 1 filter paper and vitrified by plunge freezing into a liquid ethane slurry cooled by liquid nitrogen using a manual plunger in a 4 °C cold room whose humidity was raised to 95% using a humidifier. For WalkerB mutant LONP1, LONP1 was diluted to a concentration of 2.5 mg/ml in 50 mM Tris pH 8, 75 mM KCl, 10 mM MgCl₂, 1 mM TCEP, and 1 mM ATP. For bortezomib-bound LONP1, LONP1 was diluted to a concentration of 2.5 mg/ml in 50 mM Tris pH 8, 75 mM KCl, 10 mM MgCl₂, 1 mM TCEP, and 1 mM ATPγS with the addition of 10-fold molar excess bortezomib. Samples for WalkerB and bortezomib-bound LONP1 were prepared for cryo-EM analyses using the same procedures used for the wild-type sample.

Size exclusion chromatography was performed using an Äkta Explorer (Cytiva) on a Superdex 200 Increase 10/300 GL column (Cytiva). Recombinant DFCP1 was loaded with nucleotides by incubation with either 400 µM ADP or ATPγS (Jena Bioscience) in exchange buffer (50 mM Tris, 150 mM NaCl, 50 mM MgCl₂, 10% Glycerol, 1 mM TCEP) for 20 min at RT, applied to the size exclusion column and then developed by isocratic elution with exchange buffer at 4 °C (flow rate 0.25 ml/min).