For cryo-EM analysis, the 30S particles were further purified by gel-filtration with a Superose® 6 Increase 10/300 column (GE Healthcare, Uppsala, Sweden) equilibrated with HAKM7 buffer (50 mM HEPES-KOH, pH 7.5, 70 mM NH4Cl, 30 mM KCl, 7 mM MgCl2). The 30S-containing fractions were pooled and pelleted by centrifugation in an SW 55 rotor (Beckman Coulter, Brea, CA, USA) at 50,000 rpm, for 3 h. The 30S particles were dissolved in HAKM7 buffer, aliquoted, frozen in liquid nitrogen, and stored at −70 °C. Before the application to an EM grid, wild-type 30S subunits were reactivated by a 30 min incubation at 37 °C at a high Mg2+ concentration (HAKM20, 20 mM MgCl2) and all the samples were diluted to 0.4 µM of 30S particles in HAKM7.

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