Ribosomes terminate translation when release factors (RFs) recognize an mRNA stop codon in the A (aminoacyl-tRNA) site (Brenner et al., 1967; Brenner et al., 1965). Upon stop-codon recognition, the release factor hydrolyzes peptidyl-tRNA in the P site of the large subunit, thus releasing nascent protein from the ribosome (Capecchi, 1967a; Capecchi, 1967b; Scolnick et al., 1968; Tompkins et al., 1970). In bacteria, RF1 recognizes UAA or UAG, and RF2 recognizes UAA or UGA codons (Nakamura et al., 1995). Following peptide release, RF1 or RF2 must dissociate from the ribosome to allow the ribosome and release factor to recycle. While structural studies have provided snapshots of some peptidyl-tRNA hydrolysis steps (reviewed in Dunkle and Cate, 2010; Korostelev, 2011; Ramakrishnan, 2011; Rodnina, 2018; Youngman et al., 2008), recent biophysical and biochemical findings suggest a highly dynamic series of termination events (Adio et al., 2018; Indrisiunaite et al., 2015; Prabhakar et al., 2017; Sternberg et al., 2009), which have not been structurally visualized.

In the pre-termination (pre-hydrolysis) state, the substrate peptidyl-tRNA resides in a non-rotated (classical) conformation of 70S ribosome, with its anticodon stem loop bound to the mRNA codon in the P site of the small 30S subunit and the 3′-terminal CCA-peptidyl moiety in the P site of the large 50S subunit (Agrawal et al., 2000; Ermolenko et al., 2007). Cryogenic electron microscopy (cryo-EM) and X-ray studies reveal RF1 or RF2 bound in the A site next to P tRNA (Klaholz et al., 2003; Petry et al., 2005; Rawat et al., 2006; Rawat et al., 2003), and provide insight into pre-hydrolysis (Jin et al., 2010) and post-hydrolysis states (Korostelev et al., 2008; Korostelev et al., 2010; Laurberg et al., 2008; Weixlbaumer et al., 2008). The release factor’s codon-recognition domain (domain 2) binds the stop codon in the 30S decoding center. The catalytic GGQ motif (glycine-glycine-glutamine of domain 3) inserts into the 50S peptidyl-transferase center (PTC) adjacent to the CCA end of P-tRNA. Ribosomes retain the non-rotated conformation in RF-bound structures, including those with mutated or post-translationally modified RFs (Pierson et al., 2016; Santos et al., 2013; Zeng and Jin, 2018) or when RFs are perturbed by the termination inhibitor blasticidin S (Svidritskiy and Korostelev, 2018a; Svidritskiy and Korostelev, 2018b) or by a sense codon in the A site (Svidritskiy et al., 2018). The catalytic GGQ loop adopts the same compact conformation comprising a short α-helix in pre-hydrolysis-like and post-hydrolysis structures, as discussed below.

Ribosome and RF dynamics are implicated in post-hydrolysis steps. In E. coli, RF1 dissociation is assisted by the non-essential GTPase RF3 (Freistroffer et al., 1997; Koutmou et al., 2014), whereas RF2 can spontaneously dissociate from the ribosome (Adio et al., 2018). RF3 induces rotation of the small subunit relative to the large subunit by ~9 degrees in the absence of RF1 and RF2 (Ermolenko et al., 2007; Zhou et al., 2012) and stabilizes a ‘hybrid’ state of deacylated tRNA (P/E) (Gao et al., 2007; Jin et al., 2011). In the P/E conformation, the anticodon stem loop remains in the 30SS P site, while the elbow and acceptor arm bind the L1 stalk and E (exit) site of the 50S subunit. A recent cryo-EM study (Graf et al., 2018) showed that if RF1 is locked on the post-hydrolysis ribosome by antibacterial peptide apidaecin 137 (Florin et al., 2017), the ribosome can undergo intersubunit rotation in the presence of both RF1 and RF3. By contrast, biochemical experiments showed that locking RF1 on the ribosome by inactivating its catalytic motif (GGQ->GAQ) prevented RF1 recycling by RF3 from the pre-hydrolysis ribosome (Koutmou et al., 2014), echoing the deficiency of mutant RF2 (GGQ->GAQ) in stimulating GTP hydrolysis by RF3 (Zavialov et al., 2002). These and other (Adio et al., 2018) observations indicate that peptidyl-tRNA hydrolysis is critical for RF dissociation, likely due to propensity of deacylated tRNA for exiting the P site and sampling the P/E hybrid state (Moazed and Noller, 1989) via spontaneous intersubunit rotation (Cornish et al., 2008). Nevertheless, RF3 is dispensable for growth of Escherichia coli (Grentzmann et al., 1994; Mikuni et al., 1994), and its expression is not conserved in bacteria (Leipe et al., 2002). For example, RF3 is not present in the thermophilic model organisms of the Thermus and Thermatoga genera and in infectious Chlamydiales and Spirochaetae. This means that both RF1 and RF2 are capable of performing a complete round of termination independently of RF3. Indeed, recent Förster Resonance Energy Transfer (FRET) studies demonstrate spontaneous RF1 and RF2 dissociation after peptidyl-tRNA hydrolysis (Prabhakar et al., 2017), coupled with transient RF sampling of the rotated ribosomes (Adio et al., 2018). The structural basis for RF dissociation, however, remains incompletely understood because RFs have not been structurally visualized on ribosomes with rearranged/rotated subunits in the absence of RF3 and inhibitors.

In this work, we used ensemble cryo-EM, as described earlier (Abeyrathne et al., 2016; Loveland et al., 2017), employing 3D maximum likelihood classification/sorting of particle images (Lyumkis et al., 2013; Scheres, 2010; Scheres et al., 2007) to visualize the structural dynamics of RF2 on the 70S ribosome (Figure 1, Figure 1—figure supplement 1, Figure 1—figure supplement 2 and Figure 1—figure supplement 3). The structures capture RF2 and tRNA in distinct 70S conformational states (Table 1). They reveal an unexpected rearrangement of the catalytic GGQ loop, which suggests that RF2 helps direct nascent protein out of the ribosome. Our findings reconcile biophysical observations and, together with other structural studies, allow a reconstruction of the termination course by RF2, from initial binding to dissociation.

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We formed a termination complex using E. coli 70S ribosomes and RF2 to catalyze the hydrolysis of substrate N-formyl-methionyl-tRNA^fMet (fMet-tRNA^fMet) bound to mRNA carrying an AUG codon in the P site and a UGA stop codon in the A site. Although the stop codon is placed immediately after the start codon, unlike in cellular mRNAs, such model termination complexes have been successfully used in numerous structural, biochemical and biophysical studies (Adio et al., 2018; Casy et al., 2018; Koutmou et al., 2014; Kuhlenkoetter et al., 2011; Laurberg et al., 2008; Pallesen et al., 2013; Pierson et al., 2016; Shi and Joseph, 2016; Sternberg et al., 2009). Indeed, under the conditions used for cryo-EM sample preparation in this work, RF2 supports hydrolysis of fMet-tRNA^fMet and release of fMet (Figure 1—figure supplement 4), demonstrating activity of the ribosome and RF2 in our sample. Maximum-likelihood classification of cryo-EM data in FREALIGN (Lyumkis et al., 2013) yielded five structures at average resolutions of 3.3 Å to 4.4 Å (Table 2; Figure 1). The structures differ by the degrees of intersubunit rotation and 30S head rotation (swivel), and are consistent with the following termination stages: pre-termination (i.e., no RF2; Structure I); peptidyl-tRNA hydrolysis by RF2 (Structure II); tRNA exit from the PTC (Structure III); destabilization of the catalytic domain of RF2 (Structure IV); and post-termination (i.e., after RF2 dissociates; Structure V).

Pre-termination-like Structure I contains tRNA in the P site with its 3′-CCA end positioned in the peptidyl transferase center (Figure 1A). Although the tRNA is likely partially or completely deacylated due to the action of RF2, the tRNA and ribosome conformations closely resemble those in pre-termination aminoacyl- and peptidyl-tRNA complexes (Adio et al., 2018; Cornish et al., 2008; Jin et al., 2010; Polikanov et al., 2014). The 30S subunit adopts a conformation similar to crystal structures of non-rotated ribosomes with or without RFs (Jin et al., 2010; Korostelev et al., 2008; Korostelev et al., 2006; Korostelev et al., 2010; Laurberg et al., 2008; Selmer et al., 2006; Weixlbaumer et al., 2008), and exhibits a slight ~1.5° rotation relative to the 70S•RF2 crystal structure formed with the UGA stop codon (Weixlbaumer et al., 2008).

RF2 is bound in Structures II to IV (Figure 1B–D). The 70S intersubunit rotation ranges from ~1.5° in Structure II to ~7.5° in Structure IV (Table 1). This nearly full range of rotation is typical of translocation complexes with tRNA (Agirrezabala et al., 2008; Agirrezabala et al., 2012; Dunkle et al., 2011; Fischer et al., 2010; Julián et al., 2008) and elongation factor G (EF-G) (Brilot et al., 2013; Chen et al., 2013; Frank and Agrawal, 2000; Gao et al., 2009; Pulk and Cate, 2013; Tourigny et al., 2013; Zhou et al., 2013).

The most well-resolved 3.3 Å resolution Structure II (Figure 1B) accounts for the largest population of RF2-bound ribosome particles in the sample (Table 2), consistent with stabilization of the non-rotated 30S states by RF2 observed in FRET studies (Casy et al., 2018; Prabhakar et al., 2017; Sternberg et al., 2009). The overall extended conformation of RF2 resembles that seen in published 70S•RF2 structures (Korostelev et al., 2008; Weixlbaumer et al., 2008) with its codon-recognition superdomain (domains 2 and 4; Figure 1B) bound to the UGA stop codon in the decoding center of the 30S subunit and its catalytic domain 3 inserted into the peptidyl-transferase center.

Structure II features RF2 with a dramatically rearranged catalytic loop, coinciding with the lack of density for the CCA end of the P tRNA. In previous pre- and post-hydrolysis-like structures, the P-tRNA CCA end binds the PTC, and RF2 forms a compact catalytic loop (residues 245–258). In these previous structures, the catalytic ²⁵⁰GGQ²⁵² motif resides at the tip of a short α-helix, adjacent to the terminal tRNA nucleotide A76 (Figure 2A, light shades; (Jin et al., 2010; Korostelev et al., 2008; Laurberg et al., 2008; Weixlbaumer et al., 2008). The α-helical region is held in place by A2602 of 23S ribosomal RNA (rRNA), which is critical for termination efficiency (Amort et al., 2007; Polacek et al., 2003). In Structure II, however, the catalytic loop adopts an extended β-hairpin conformation that reaches ~10 Å deeper into the peptide tunnel (Figure 2A–B). To accommodate the extended β-hairpin, nucleotides G2505 and U2506 of 23S rRNA are shifted to widen the PTC (Figure 2—figure supplement 1A). The center of the β-hairpin occupies the position normally held by A76 of P tRNA (Figure 2A). The lack of density for the CCA moiety thus suggests that the 3′ end of P tRNA is at least partially released from the PTC. Indeed, formation of the β-hairpin and release of the CCA moiety is accompanied by a slight shift in the P-tRNA acceptor arm away from the PTC and by ~160° rotation of nucleotide A2602 to form a Hoogsteen base pair with C1965 at helix 71 of 23S rRNA (Figure 2A and Figure 2Figure 2—figure supplement 1B). The cryo-EM map shows strong density for the extended β strands (Figure 2C), but weaker density for the GGQ residues at the tip of the hairpin, consistent with conformational heterogeneity of the flexible glycine backbone (Figure 2—figure supplement 2). Nevertheless, the tip of the β-hairpin plugs the narrowest region of the peptide tunnel at A2062 of 23S rRNA (Figure 2B). Thus, Structure II appears to represent a previously unseen post-peptide-release state with deacyl-tRNA dissociating from the PTC.

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In Structure III (Figure 1C), a 5° rotation of the 30S subunit shifts the tRNA elbow by ~10 Å along protein L5 toward the E site. As a result, the P-tRNA acceptor arm pulls further out of the 50S P site, and the end of the acceptor arm helix points at helix α7 of RF2 near positively charged Lys 282 and Lys 289. The CCA moiety remains unresolved. The 5° intersubunit rotation also causes a slight 2° rotation of RF2 domain 3 relative to domain 2, but the overall conformation of RF2 remains similar to that in Structure II, with the tip of the extended β-hairpin plugging the peptide exit tunnel.

In Structure IV, the 30S subunit is rotated 7.5° and the P-tRNA elbow is bound to the L1 stalk, an interaction normally observed with the P/E or E tRNA (Dunkle et al., 2011; Korostelev et al., 2006; Selmer et al., 2006). Unlike P/E tRNA, however, the acceptor arm is oriented between the 50S P and E sites (Figure 1D), suggesting that the tRNA adopts a transient state on its way toward the hybrid P/E conformation. This transition coincides with an ~3° rotation (swivel) of the 30S head, characteristic of intermediate stages of tRNA and mRNA translocation (Abeyrathne et al., 2016; Ratje et al., 2010). The codon-recognition superdomain of RF2 in its canonical conformation moves with the 30S subunit, causing the catalytic domain to shift by ~5 Å from its position in Structure II (Figure 3A). Indeed, reduced density for the catalytic domain and domain 1, which bridges the 30S and 50S subunits at the A site periphery (Figure 1B), suggests that intersubunit rotation has destabilized RF2. In Structure IV, therefore, RF2 appears to be poised for dissociation and collapsing toward the compact conformation adopted by free RFs (Figure 3A) (JCSG, 2005; Shin et al., 2004; Vestergaard et al., 2001; Zoldák et al., 2007).

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Structure V is incompatible with RF2 binding and provides further insights into RF2 dissociation. The rotated 70S•tRNA structure resembles hybrid P/E states, with the acceptor arm of tRNA directed toward the E site of 50S (Figure 1E). However, the central intersubunit bridge B2a—formed by helix 69 (H69, nt. 1906–1924) of 23S rRNA—is disassembled (Figure 3B–C). In RF-bound structures (including Structures I to IV, above), the tip of H69 (at nt 1913) locks into the decoding center in a termination-specific arrangement (Korostelev et al., 2008; Korostelev et al., 2010; Laurberg et al., 2008; Weixlbaumer et al., 2008). H69 interacts with the switch loop of RFs, where the codon-recognition superdomain connects to catalytic domain 3 (e.g., Structure II, Figure 3B). This interaction directs the catalytic domain toward the PTC (Korostelev et al., 2010; Laurberg et al., 2008) and thus defines the efficiency and accuracy of release factors (Svidritskiy and Korostelev, 2018a). In Structure V, however, H69 is disengaged from the decoding center and moved ~15 Å toward the large subunit to pack against H71 near A2602 (nt 1945–1961; Figure 3C). The new position of H69 would clash with extended or compact conformations of RF2 (Figure 3—figure supplement 1), if the release factor had remained bound to the decoding center.

Release of RF2 also appears to be coupled to rotation (swivel) of the 30S head domain. In RF2-bound structures, the conserved codon-recognition ²⁰⁵SPF²⁰⁷ motif (serine-proline-phenylalanine of domain 2) of RF2 interacts with the stop codon and the 30S head near bulged nucleotide C1054 of 16S rRNA. In Structure V, a 6° rotation of the 30S head shifts the position of C1054 by 5 Å from its position in RF2-bound Structure IV, consistent with disassembly of RF2 contacts. The disruption of decoding-center interactions due to repositioning of H69 and head swivel therefore explain the absence of RF2 in Structure V.

Mechanism of translation termination by RF2

Our termination structures allow us to reconstruct a dynamic pathway for termination (Figure 4 and Animation 1) and reconcile biophysical and biochemical findings (Adio et al., 2018; Casy et al., 2018; Prabhakar et al., 2017; Sternberg et al., 2009). Recent structural studies revealed compact RF conformations on non-rotated 70S ribosomes (Fu et al., 2018; Svidritskiy and Korostelev, 2018a) that suggest how RFs open at early stages of codon recognition (He and Green, 2010; Hetrick et al., 2009; Trappl and Joseph, 2016). The short-lived transition from compact to open conformation(s) lasts tens of milliseconds (Fu et al., 2018), and compact RF2 conformations were not captured in our sample. Compact RF2 was also visualized by cryo-EM on the 70S ribosome in the presence of truncated mRNA and alternative rescue factor A (ArfA) (Demo et al., 2017; James et al., 2016), suggesting a conserved pathway for initial binding of RFs to non-rotated ribosomes. Together with these recent studies, our work provides an expanded structural view of the termination reaction – from initial codon recognition to peptide release to RF2 dissociation (Figure 4).

Figure 4

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Termination begins when release factor recognizes a stop codon in the A site of a non-rotated (or slightly rotated) 70S ribosome carrying peptidyl-tRNA (Figure 4A). A compact release factor recognizes the stop codon, while its catalytic domain is kept 60 to 70 Å away from the PTC (Fu et al., 2018). Separation of the codon-recognition and catalytic functions during initial binding of RF underlies high accuracy of termination (He and Green, 2010; Laurberg et al., 2008; Svidritskiy and Korostelev, 2018a; Trappl and Joseph, 2016). Upon binding the decoding center, steric hindrance with P-tRNA forces the RF catalytic domain to undock from domain two and sample the intersubnit space (Figure 4B) (discussed in Svidritskiy and Korostelev, 2018a). The ribosome remains non-rotated because peptidyl-tRNA bridges the P sites of 30S and 50S subunits. Interactions between the switch loop of RF and the decoding center and H69 direct the catalytic domain into the PTC. The GGQ catalytic loop is placed next to the scissile ester bond of peptidyl-tRNA and stabilized by A2602 (Jin et al., 2010; Laurberg et al., 2008), so that the Gln backbone amide of the GGQ motif can catalyze peptidyl-tRNA hydrolysis (Figure 4C) (Korostelev et al., 2008; Santos et al., 2013). These ribosome and RF2 rearrangements complete the first stage of termination leading to peptidyl-tRNA hydrolysis—an irreversible step that separates the newly made protein from P-site tRNA and prevents further elongation.

To be recycled, the ribosome must release the nascent protein and RF. The deacylated CCA end of the tRNA exits the 50S P site, which allows movement of A2602. No longer stabilized by the tRNA and A2602, the GGQ loop reorganizes into an extended β-hairpin that blocks the peptide tunnel (Structure II), thus biasing the diffusion of nascent peptide toward the exit of the peptide tunnel at the surface of the 50S subunit (Figure 4D). Furthermore, the exit of the CCA end from the P site supports formation of the P/E-tRNA hybrid conformation via spontaneous intersubunit rotation (Structures III and IV), which is also essential for translocation of tRNA-mRNA (reviewed in Ling and Ermolenko, 2016; Noller et al., 2017). The large counter-clockwise rotation of the small subunit destabilizes the catalytic domain of RF2 (Structure IV), so that it begins to collapse toward domain 2 bound to the 30S subunit (Figure 4E). Rotation of the 30S head and detachment of H69 from the decoding center force dissociation of RF2 from the A site of the 30S subunit (Structure V, Figure 4F).

Remarkably similar extents of intersubunit rotation and head rotation were observed in recent RF1-bound 70S structures formed with RF3•GDPNP in the presence of apidaecin (which stalls RF1 on the ribosome). In the presence of stalled RF1, however, the intersubunit bridge remains attached to the decoding center (Graf et al., 2018). Thus, the ribosome samples globally similar rotation states with RF1 and RF2, with or without RF3. RF3 stabilizes the rotated state (Ermolenko et al., 2007; Gao et al., 2007; Jin et al., 2011; Zhou et al., 2012) to promote RF release. However, the role of RF3 in termination is dispensable because intersubunit rotation is an inherent— that is spontaneous and thermally driven—property of the ribosome (Cornish et al., 2008). Cold sensitivity of RF3-deletion strains (Nichols et al., 2011; O'Connor, 2015) perhaps manifests the dependence on RF3 due to perturbation of the intersubunit dynamics at lower temperatures. In the fully rotated state, the post-termination ribosome becomes a substrate for subunit dissociation by EF-G and recycling factor RRF (Agrawal et al., 2004; Dunkle et al., 2011; Fu et al., 2016; Prabhakar et al., 2017). Structure V is consistent with structural studies showing ribosome recycling factor (RRF) bound to ribosomes with a dislodged intersubunit bridge B2a (Borovinskaya et al., 2007; Pai et al., 2008), which helps split the ribosome into subunits. Translation termination and recycling of the release factors and the ribosome therefore rely on the spontaneous ribosome dynamics (Adio et al., 2018; Cornish et al., 2008; Ermolenko et al., 2007; Prabhakar et al., 2017; Sternberg et al., 2009), coupled with local rearrangements of the universally conserved elements of the peptidyl-transferase and decoding centers.

Structural models have been deposited in PDB under the accession codes 6OFX, 6OG7, 6OGF, 6OGG, 6OGI. Cryo-EM data have been deposited to EMDB under the accession codes EMD-20048, EMD-20052, EMD-20056, EMD-20057, EMD-20058.

1. 1. Svidritskiy E
   2. Demo G
   3. Loveland AB
   4. Xu C
   5. Korostelev AA

   (2019) Protein Data Bank

   ID 6OFX. Non-rotated ribosome (Structure I).

   http://www.rcsb.org/structure/6OFX
2. 1. Svidritskiy E
   2. Demo G
   3. Loveland AB
   4. Xu C
   5. Korostelev AA

   (2019) Electron Microscopy Data Bank

   ID EMD-20048. Non-rotated ribosome (Structure I).

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-20048
3. 1. Svidritskiy E
   2. Demo G
   3. Loveland AB
   4. Xu C
   5. Korostelev AA

   (2019) Protein Data Bank

   ID 6OG7. 70S termination complex with RF2 bound to the UGA codon. Non-rotated ribosome with RF2 bound (Structure II).

   http://www.rcsb.org/structure/6OG7
4. 1. Svidritskiy E
   2. Demo G
   3. Loveland AB
   4. Xu C
   5. Korostelev AA

   (2019) Protein Data Bank

   ID 6OGF. 70S termination complex with RF2 bound to the UGA codon. Partially rotated ribosome with RF2 bound (Structure III).

   http://www.rcsb.org/structure/6OGF
5. 1. Svidritskiy E
   2. Demo G
   3. Loveland AB
   4. Xu C
   5. Korostelev AA

   (2019) Protein Data Bank

   ID 6OGI. 70S termination complex with the UGA stop codon. Rotated ribosome conformation (Structure V).

   http://www.rcsb.org/structure/6OGI
6. 1. Svidritskiy E
   2. Demo G
   3. Loveland AB
   4. Xu C
   5. Korostelev AA

   (2019) Protein Data Bank

   ID 6OGG. 70S termination complex with RF2 bound to the UGA codon. Rotated ribosome with RF2 bound (Structure IV).

   http://www.rcsb.org/structure/6OGG
7. 1. Svidritskiy E
   2. Demo G
   3. Loveland AB
   4. Xu C
   5. Korostelev AA

   (2019) Electron Microscopy Data Bank

   ID EMD-20052. 70S termination complex with RF2 bound to the UGA codon. Non-rotated ribosome with RF2 bound (Structure II).

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-20052
8. 1. Svidritskiy E
   2. Demo G
   3. Loveland AB
   4. Xu C
   5. Korostelev AA

   (2019) Electron Microscopy Data Bank

   ID EMD-20056. 70S termination complex with RF2 bound to the UGA codon. Partially rotated ribosome with RF2 bound (Structure III).

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-20056
9. 1. Svidritskiy E
   2. Demo G
   3. Loveland AB
   4. Xu C
   5. Korostelev AA

   (2019) Electron Microscopy Data Bank

   ID EMD-20057. 70S termination complex with RF2 bound to the UGA codon. Rotated ribosome with RF2 bound (Structure IV).

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-20057
10. 1. Svidritskiy E
    2. Demo G
    3. Loveland AB
    4. Xu C
    5. Korostelev AA

    (2019) Electron Microscopy Data Bank

    ID EMD-20058. 70S termination complex with the UGA stop codon. Rotated ribosome conformation (Structure V).

    https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-20058

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Author details

1. Egor Svidritskiy

   RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—review and editing, Assembled the ribosome complexes, Performed biochemistry, Collected and processed cryo-EM data, Modeled and refined structural models

   Competing interests

   No competing interests declared

2. Gabriel Demo

   RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Purified ribosome subunits and RF2, Assisted with structure refinements

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5472-9249

3. Anna B Loveland

   RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States

   Contribution

   Formal analysis, Methodology, Assisted with data processing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9172-7747

4. Chen Xu

   Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States

   Contribution

   Methodology, Implemented multiple-shot (per hole) data acquisition, Optimized data collection

   Competing interests

   No competing interests declared

5. Andrei A Korostelev

   1. RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, United States
   2. Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, United States

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Writing—original draft, Project administration, Writing—review and editing, Supervised the project, Assisted with modeling and structure refinements, Wrote the manuscript with input from co-authors

   For correspondence

   andrei.korostelev@umassmed.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1588-717X

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