Ja 25.50 motor

Recombinant RAC was purified as described in our previous work^{37 (link)}. Coding fragment of Ssb1 was amplified from S. cerevisiae genomic DNA (S288C), and cloned into the vector pET28a. The plasmid was transformed into Escherichia coli BL21 (DE3) cells for overexpression. Cells were induced at 18 °C with 0.4 mM IPTG for 12 h, harvested, resuspended in buffer A (20 mM HEPES-KOH pH 7.5, 500 mM KCl, 10 mM MgCl₂, 1 mM PMSF, and 20 mM imidazole) and subjected to ultrasonic lysis. The cell lysates were then clarified by centrifugation at 30,970 x g for 30 min at 4 °C in a JA 25.50 motor (Beckman Coulter). Supernatants were loaded onto a Ni-NTA column (GE Healthcare) and eluted with buffer B (20 mM HEPES-KOH pH 7.5, 120 mM KCl, 10 mM MgCl₂, and 250 mM imidazole). Eluates were further purified with a Resource Q column (1 ml, GE Healthcare). The Ssb1-containing fractions were pooled, concentrated and loaded onto a pre-equilibrated Superdex 200 column (10/300 GL, GE Healthcare) with buffer C (20 mM HEPES-KOH pH 7.5, 100 mM KCl, 10 mM MgCl₂).

Cells were grown in 4 L YPD medium to an OD₆₀₀ of 2.0, harvested and washed with 50 ml ice-cold lysis buffer (40 mM HEPES-KOH pH 7.5, 100 mM KCl, 10 mM MgCl₂). Cell pellet was resuspended with lysis buffer of equal volume supplied with 0.05% NP40 and protease inhibitor cocktail (cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpack, Roche) and disrupted by vortexing with acid-washed glass beads in 50 ml tube. Cell lysate was clarified by centrifugation at 30,970 x g for 45 min at 4 °C in a JA 25.50 motor (Beckman Coulter) and the supernatant was incubated with pre-equilibrated Anti-FLAG M2 Affinity Gel (Sigma) for 90 min at 4 °C. The beads were then washed with 10 ml lysis buffer for three times and the samples were competitively eluted with 100 μg/ml flag peptides dissolved in lysis buffer for 30 min at 4 °C. The elution was concentrated with Amicon Ultra centrifugal filter units (Millipore) at 3000 g to an A₂₆₀ of ~12 for cryo-EM grid preparation. Samples were also examined by SDS-PAGE, and selected bands were subjected to mass spectrometry (Supplementary Fig. 1a).