Saccharomyces cerevisiae strain SCCM49 (MATa; his3Δ1; leu2Δ0; met15Δ0, ura3Δ0; YLR384C-3xFLAG::natNT2), the Elp1-3xFLAG strain, was generated by integrating a cassette of the 3xFLAG tag and a natNT2 marker into the genome of the parental strain BY4741. Yeast cells were grown as described previously (26). Cells (350 to 400 g) expressing C terminally FLAG-tagged Elp1 were suspended in buffer A [250 mM Hepes (pH 7.9), 125 mM NaCl, 0.1% NP-40, 10% glycerol, 50 mM NaF, 0.1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF)] supplemented with protease inhibitor cocktail (cOmplete EDTA-free, Roche) and lysed at 4°C with glass beads in BeadBeater (BioSpec). The soluble fraction obtained after centrifugation (1 hour at 30,000g in a Beckman JA-14 rotor) was incubated with 5 ml of preequilibrated ANTI-FLAG M2 affinity gel (Sigma-Aldrich) for 2 to 3 hours at 4°C. After washing the M2 resin with buffer B [10 mM Hepes (pH 7.9), 125 mM NaCl, 0.1% NP-40, 10% glycerol, 50 mM NaF, 0.1 mM Na3VO4, 1 mM PMSF supplemented with protease inhibitor cocktail] and buffer C [10 mM Hepes (pH 7.9), 125 mM NaCl, 10% glycerol, 50 mM NaF, 0.1 mM Na3VO4], bound Elp123 was eluted in buffer C containing 3× FLAG peptide (0.1 mg/ml; Sigma-Aldrich). A final concentration of 5 mM 2-mercaptoethanol was added to the eluate. The sample was concentrated to 7 to 20 mg/ml and subjected to a final purification step using a Superose 6 Increase 3.2/300 column (GE Healthcare) in buffer D [10 mM Hepes (pH 7.9), 125 mM NaCl, 50 mM NaF, 0.1 mM Na3VO4, 5 mM 2-mercaptoethanol] or buffer E [10 mM Hepes (pH 7.9), 125 mM NaCl, 50 mM NaF, 0.1 mM Na3VO4, 5 mM dithiothreitol (DTT), 5 mM MgCl2]. For the apoElp123 reconstruction, the gel filtration was performed in buffer D. Purified Elp123 was incubated with in vitro–transcribed S. cerevisiae tRNAAlaUGC at a 1:3 molar ratio for 30 min at 25°C in the presence of 1 mM MgCl2 and 1 mM acetyl-CoA. The sample was cross-linked with 0.01% glutaraldehyde (Electron Microscopy Sciences) for 1 hour at 4°C, quenched with 40 mM tris-HCl buffer (final concentration) for 10 min, and then plunged. Following tRNA binding, experiments showed that Elp123 binding to tRNA was abolished in the presence of 0.01% glutaraldehyde (fig. S4A), validating the observation of apoElp123 but not Elp123-tRNA complexes in the cross-linked sample. Hence, for the Elp123-tRNA complexes, pure Elp123 (from gel filtration in buffer E) was incubated with in vitro–transcribed tRNAAlaUGC at a 1:5 molar ratio for 30 min at 25°C (in the presence of 1 mM acetyl-CoA for the Elp123-tRNA lobe dataset and without acetyl-CoA for the Elp123-2tRNA dataset) and then immediately plunged.

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