a, Final resolution of the Vms1(∆VIM/Q295L) pre-state in the presence of Arb1. b, Local resolution map of the pre-state in the presence of Arb1. Left, overview; right, transverse view. The electron density is displayed at 2.21σ. c, Local resolution map of Arb1 with peptidyl-tRNA. d, Density and docked models of Arb1 and tRNA in yellow and blue, respectively. The tRNA density in c, d is displayed at 3.59σ and the rest remains at 2.21σ. e, View of the Arb1 model surface in two orientations. The nucleotide binding domains (NBD1 and 2) and the LD are indicated. f, Comparison of Arb1 with ABCF-type ATPases in open and closed states. The models of the ADP-bound RLI from P. furiosus (PDB 1YQT), the AMP-PNP bound Rli1 from S. cerevisiae (PDB 5LL6) and the AMP-PNP bound MsrE from Pseudomonas aeruginosa (PDB 5ZLU) were compared to Arb1 by rigid-body fitting of NBD1 into the density of the Arb1 NBD1. g, Steric clash of Arb1–tRNA Vms1 with Rqc2–P-tRNA Rqc2 on the 60S subunit. Arb1, tRNA Vms1 and Vms1 LRS–VLRF1 domain are shown as models and Rqc2, A-tRNA Rqc2 and P-tRNA Rqc2 as densities. See Methods for the contour levels. h, Overexpression of Arb1–Myc does not lead to displacement of Rqc2 on 60S subunits. Lysates of cells expressing Rqc2–3HA from its endogenous promoter with or without overexpression of Arb1–1×Myc were analysed by sucrose-gradient centrifugation. Fractions were analysed by SDS–PAGE and immunoblotting using HA and Myc antibodies. 60S and 80S ribosomes were detected using anti-Rpl3 antibody. T, top and B, bottom of the gradient. i, Density and molecular model of Vms1, tRNA Vms1 , uL16 and Arb1 of the Vms1–60S pre-state in the presence of Arb1. The density of 25S A2971 is displayed at 5.07σ, Arb1 at 4.55σ, Vms1 at 4.34σ, tRNA at 3.87σ and uL16 at 3.76σ. j, Model illustrating the rotation of 25S A2971 (A2602 in E. coli) by 180° upon Arb1 binding. k, The vms1∆ arb1∆ shuffle strain was complemented either with an Arb1(WT) construct or an Arb1-sAid-HA degron plasmid (Arb1::degron). Growth on YPD and YPD + auxin (500 µM final concentration) plates was monitored after two days at 30 °C (left). Arb1 protein level of the vms1∆ arb1::degron strain before and after treatment with auxin (500 µM final concentration) for 60 min at 30 °C (right). Cell lysates were analysed by immunoblotting using the HA antibody. l, Yeast in vitro release reaction of arrested peptides translated from the NS-3×Flag RQC reporter mRNA in vms1∆ski2∆ lysate. Buffer (−), Arb1, Vms1 or Vms1 together with Arb1 were added to cycloheximide-stopped translation reactions. Incubation time: 5 min (lane 1 and 2) at 25 °C; 0, 2 or 5 min, respectively (lanes 3–12). Molar ratios of Vms1 and Arb1 are 1 to 25 (Vms1 + Arb1) and 1 to 50 (Vms1 1/5 + Arb1), respectively. Bottom, longer exposure of the relatively weak bands. CCA, CCA from tRNA 3′ end; pep: peptidyl or peptide; * and ** indicate peptidyl-tRNA and free peptide from the colliding ribosome, respectively. m, Overexpression of Arb1 suppresses aggregation of a NS-cGFP reporter construct. Cell extracts of ltn1∆ cells expressing NS-cGFP and overexpressing Myc-tagged Arb1 or indicated Arb1 mutants were analysed by immunoblotting using anti-GFP and anti-Myc antibodies. n, Cell lysates of vms1∆arb1∆ cells expressing Arb1–HA or indicated Arb1 mutants without or with expression of Vms1–3×Myc were analysed by immunoprecipitation using Myc antibody. The inputs and the immunoprecipitates were analysed by Coomassie brilliant blue staining and immunoblotting using HA antibody. Asterisks indicate IgG heavy and light chains. o, Densities and molecular models of Vms1, tRNA Vms1 and uL16 loop in the two pre-states either without Arb1 (left) or with Arb1 (middle). Right, the superposition of the uL16 loop in the Arb1 containing pre-state and in the eIF5A-bound 60S ribosomal subunit (PDB 5GAK) in purple and pink, respectively. The density of uL16 left structure is displayed at 4.06σ; the rest remains the same as in i. p, Model of tRNA positioning and dislocation by Arb1. See Supplementary Fig. 1 for gel source images.