Quantifoil grids (200 mesh 2/1 copper) were glow-discharged on a PELCO easiGlow glow discharger. A total of 2.5 μl of sample (0.37 to 0.4 mg/ml) was plunge-frozen using FEI Vitrobot Mark IV set to 100% humidity and 4°C (blotting parameters: wait time, 15 s; blot force, 5; blot time, 5 to 8 s). Micrographs were acquired at 300 kV using FEI Titan Krios equipped with a Gatan Quantum energy filter and a K2 Summit direct detector. The detector was operated in super-resolution mode at ×105,000 magnification, resulting in a pixel size of 0.675 Å on the object scale. A total of 4614 micrographs were collected for the apoElp123 reconstruction, 6990 for the Elp123-tRNA lobe reconstruction, and 5412 for the Elp123-2tRNAs reconstruction. We collected at under-focus varying between 1 and 2.5 μm a total of 40 frames accumulating to doses of 43 e2 (apoElp123), 44 e2 (Elp123-tRNA lobe), and 39.8 e2 (Elp123-2tRNAs).

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