Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome

Virus propagation relies on the host translational apparatus. To efficiently compete with host mRNAs and engage in translation under stress, some viral mRNAs undergo cap-independent translation. To this end, internal ribosome entry site (IRES) RNAs are employed (reviewed in Deforges et al. (2015), Jackson et al. (2010), Lozano and Martinez-Salas (2015). An IRES is located at the 5’ untranslated region of the viral mRNA, preceding an open reading frame (ORF). To initiate translation, a structured IRES RNA interacts with the 40S subunit or the 80S ribosome, resulting in precise positioning of the downstream start codon in the small 40S subunit. The canonical scenario of cap-dependent and IRES-dependent initiation involves positioning of the AUG start codon and the initiator tRNA^Met in the ribosomal peptidyl-tRNA (P) site, facilitated by interaction with initiation factors (Jackson et al., 2010). Subsequent binding of an elongator aminoacyl-tRNA to the ribosomal A site transitions the initiation complex into the elongation cycle of translation. Upon peptide bond formation, the two tRNAs and their respective mRNA codons translocate from the A and P to P and E (exit) sites, freeing the A site for the next elongator tRNA.

An unusual strategy of initiation is used by intergenic-region (IGR) IRESs found in Dicistroviridae arthropod-infecting viruses. These include shrimp-infecting Taura syndrome virus (TSV; Cevallos and Sarnow, 2005; Hatakeyama et al., 2004), and insect viruses Plautia stali intestine virus (PSIV; Nishiyama et al. (2003), Sasaki and Nakashima (1999)) and Cricket paralysis virus (CrPV; Jan et al. (2001), Wilson et al. (2000)). The IGR IRES mRNAs do not contain an AUG start codon. The IGR-IRES-driven initiation does not involve initiator tRNA^Met and initiation factors (Jan et al., 2001; Sasaki and Nakashima, 1999; Wilson et al., 2000). As such, this group of IRESs represents the most streamlined mechanism of eukaryotic translation initiation. A recent demonstration of bacterial translation initiation by an IGR IRES (Colussi et al., 2015) indicates that the IRESs take advantage of conserved structural and dynamic properties of the ribosome. Early electron cryo-microscopy (cryo-EM) studies have found that the CrPV IRES packs in the ribosome intersubunit space (Schuler et al., 2006; Spahn et al., 2004b). Recent cryo-EM structures of ribosome-bound TSV IRES (Koh et al., 2014) and CrPV IRES (Fernandez et al., 2014) revealed that IGR IRESs position the ORF by mimicking a translating ribosome bound with tRNA and mRNA. The ~200-nt IRES RNAs span from the A site beyond the E site. A conserved tRNA-mRNA–like structural element of pseudoknot I (PKI; Costantino et al., 2008) interacts with the decoding center in the A site of the 40S subunit (Fernandez et al., 2014; Koh et al., 2014). The codon-anticodon-like helix of PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 (G530, A1492 and A1493 in E. coli 16S ribosomal RNA, or rRNA). The downstream initiation codon—coding for alanine—is placed in the mRNA tunnel, preceding the decoding center. PKI of IGR IRESs therefore mimics an A-site elongator tRNA interacting with an mRNA sense codon, but not a P-site initiator tRNA^Met and the AUG start codon.

How this non-canonical initiation complex transitions to the elongation step is not fully understood. For a cognate aminoacyl-tRNA to bind the first viral mRNA codon, PKI has to be translocated from the A site, so that the first codon can be presented in the A site. A cryo-EM structure of the ribosome bound with a CrPV IRES and release factor eRF1 occupying the A site provided insight into the post-translocation state (Muhs et al., 2015). In this structure, PKI is positioned in the P site and the first mRNA codon is located in the A site. How the large IRES RNA translocates within the ribosome, allowing PKI translocation from the A to P site is not known.

The structural similarity of PKI and the tRNA anticodon stem loop (ASL) bound to a codon suggests that their mechanisms of translocation are similar to some extent. Translocation of the IRES or tRNA-mRNA requires eukaryotic elongation factor 2 (eEF2) (Jan et al., 2003; Pestova and Hellen, 2003), a structural and functional homolog of the well-studied bacterial EF-G (Czworkowski et al., 1994; Evarsson et al., 1994). Pre-translocation tRNA-bound ribosomes contain a peptidyl- and deacyl-tRNA, both base-paired to mRNA codons in the A and P sites (termed 2tRNA•mRNA complex). Translocation of 2tRNA•mRNA involves two major large-scale ribosome rearrangements (Figure 1—figure supplement 1) (reviewed in Ling and Ermolenko, (2016)). First, studies of bacterial ribosomes showed that a ~10° rotation of the small subunit relative to the large subunit, known as intersubunit rotation, or ratcheting (Frank and Agrawal, 2000), is required for translocation (Horan and Noller, 2007). Intersubunit rotation occurs spontaneously upon peptidyl transfer, and is coupled with formation of hybrid tRNA states (Agirrezabala et al., 2008; Blanchard et al., 2004; Cornish et al., 2008; Ermolenko et al., 2007; Julián et al., 2008; Moazed and Noller, 1989). In the rotated pre-translocation ribosome, the peptidyl-tRNA binds the A site of the small subunit with its ASL and the P site of the large subunit with the CCA 3’ end (A/P hybrid state). Concurrently, the deacyl-tRNA interacts with the P site of the small subunit and the E site of the large subunit (P/E hybrid state). The ribosome can undergo spontaneous, thermally-driven forward-reverse rotation (Cornish et al., 2008) that shifts the two tRNAs between the hybrid and 'classical' states while the anticodon stem loops remain non-translocated. Binding of EF-G next to the A site and reverse rotation of the small subunit results in translocation of both ASLs on the small subunit (Ermolenko and Noller, 2011). EF-G is thought to 'unlock' the pre-translocation ribosome (Savelsbergh et al., 2003; Spirin, 1969), allowing movement of the 2tRNA•mRNA complex, however the structural details of this unlocking are not known.

The second large-scale rearrangement involves rotation, or swiveling, of the head of the small subunit relative to the body. The head can rotate by up to ~20° around the axis nearly orthogonal to that of intersubunit rotation, in the absence of tRNA (Schuwirth et al., 2005) or in the presence of a single P/E tRNA and eEF2 (Taylor et al., 2007) or EF-G (Ratje et al., 2010). Förster resonance energy transfer (FRET) data suggest that head swivel of the rotated small subunit facilitates EF-G-mediated movement of 2tRNA•mRNA (Guo and Noller, 2012). Structures of the 70S•EF-G complex bound with two nearly translocated tRNAs (Ramrath et al., 2013; Zhou et al., 2014), exhibit a large 18° to 21° head swivel in a mid-rotated subunit, whereas no head swivel is observed in the fully rotated pre-translocation or in the non-rotated post-translocation 70S•2tRNA•EF-G structures (Brilot et al., 2013; Gao et al., 2009). The structural role of head swivel is not fully understood. The head swivel was proposed to facilitate transition of the tRNA from the P to E site by widening a constriction between these sites on the 30S subunit (Schuwirth et al., 2005). This widening allows the ASL to sample positions between the P and E sites (Ratje et al., 2010). Whether and how the head swivel mediates tRNA transition from the A to P site remains unknown.

We sought to address the following questions by structural visualization of 80S•IRES•eEF2 translocation complexes: (1) How does a large IRES RNA move through the restricted intersubunit space, bringing PKI from the A to P site of the small subunit? (2) How does eEF2 mediate IRES translocation? (3) Does IRES translocation involve large rearrangements in the ribosome, similar to tRNA translocation? (4) What, if any, is the mechanistic role of 40S head rotation in IRES translocation? We used cryo-EM to visualize 80S•TSV IRES complexes formed in the presence of eEF2•GTP and the translation inhibitor sordarin, which stabilizes eEF2 on the ribosome. Although the mechanism of sordarin action is not fully understood, the inhibitor does not affect the conformation of eEF2•GDPNP on the ribosome (Taylor et al., 2007), rendering it an excellent tool in translocation studies. Maximum-likelihood classification using FREALIGN (Lyumkis et al., 2013) identified five IRES-eEF2-bound ribosome structures within a single sample (Figures 1 and 2). The structures differ in the positions and conformations of ribosomal subunits (Figures 1b and 2), IRES RNA (Figures 3 and 4) and eEF2 (Figures 5 and 6). This ensemble of structures allowed us to reconstruct a sequence of steps in IRES translocation induced by eEF2.

Figure 1 with 3 supplements see all

Download asset Open asset

Figure 1—source data 1

Structure refinement statistics for Structures I, II, III, IV, V.

https://doi.org/10.7554/eLife.14874.003

Download elife-14874-fig1-data1-v2.pdf

We used single-particle cryo-EM and maximum-likelihood image classification in FREALIGN to obtain three-dimensional density maps from a single specimen. The translocation complex was formed using S. cerevisiae 80S ribosomes, Taura syndrome virus IRES, and S. cerevisiae eEF2 in the presence of GTP and the eEF2-binding translation inhibitor sordarin. Unsupervised cryo-EM data classification was combined with the use of three-dimensional and two-dimensional masking around the ribosomal A site (Figure 1—figure supplement 2). This approach revealed five 80S•IRES•eEF2•GDP structures at average resolutions of 3.5 to 4.2 Å, sufficient to locate IRES domains and to resolve individual residues in the core regions of the ribosome and eEF2 (Figures 3c,d, and 5f,h; see also Figure 1—figure supplement 2 and Figure 5—figure supplement 2), including the post-translational modification diphthamide 699 (Figure 3c).

Our structures represent hitherto uncharacterized translocation complexes of the TSV IRES captured within globally distinct 80S conformations (Figures 1b and 2). We numbered the structures from I to V, according to the position of the tRNA-mRNA-like PKI on the 40S subunit (Figure 2—source data 1). Specifically, PKI is partially withdrawn from the A site in Structure I, and fully translocated to the P site in Structure V (Figure 4; see also Figure 3—figure supplement 1). Thus Structures I to IV represent different positions of PKI between the A and P sites (Figure 2—source data 1), suggesting that these structures describe intermediate states of translocation. Structure V corresponds to the post-translocation state.

Figure 2 with 1 supplement see all

Download asset Open asset

Figure 2—source data 1

Measurements for conformations and positions in Structures I through V.

https://doi.org/10.7554/eLife.14874.008

Download elife-14874-fig2-data1-v2.pdf

Changes in ribosome conformation and eEF2 positions are coupled with IRES movement through the ribosome

Intersubunit rotation

Using the post-translocation S. cerevisiae 80S ribosome bound with the P and E site tRNAs as a reference (80S•2tRNA•mRNA), in which both the subunit rotation and the head-body swivel are 0°(Svidritskiy et al., 2014), we found that the ribosome adopts four globally distinct conformations in Structures I through V (Figure 1b; see also Figure 1—figure supplement 1 and Figure 2—source data 1). Structure I comprises the most rotated ribosome conformation (~10°), characteristic of pre-translocation hybrid-tRNA states. From Structure I to V, the body of the small subunit undergoes backward (reverse) rotation (Figure 2b; see also Figure 1—figure supplement 2 and Figure 2—figure supplement 1). Structures II and III are in mid-rotation conformations (~5°). Structure IV adopts a slightly rotated conformation (~1°). Structure V is in a nearly non-rotated conformation (0.5°), very similar to that of post-translocation ribosome-tRNA complexes (Gao et al., 2009; Korostelev et al., 2006; Selmer et al., 2006; Svidritskiy et al., 2014). Thus, intersubunit rotation of ~9° from Structure I to V covers a nearly complete range of relative subunit positions, similar to what was reported for tRNA-bound yeast (Svidritskiy et al., 2014; Taylor et al., 2007), bacterial (Agirrezabala et al., 2008; Fischer et al., 2010; Julián et al., 2008) and mammalian (Budkevich et al., 2011) ribosomes.

40S head swivel

The pattern of 40S head swivel between the structures is different from that of intersubunit rotation (Figures 2c and d; see also Figure 2—source data 1). As with the intersubunit rotation, the small head swivel (~1°) in the non-rotated Structure V is closest to that in the 80S•2tRNA•mRNA post-translocation ribosome (Svidritskiy et al., 2014). However in the pre-translocation intermediates (from Structure I to IV), the beak of the head domain first turns toward the large subunit and then backs off (Figure 2—figure supplement 1). This movement reflects the forward and reverse swivel. The head samples a mid-swiveled position in Structure I (12°), then a highly-swiveled position in Structures II and III (17°) and a less swiveled position in Structure IV (14°). The maximum head swivel is observed in the mid-rotated complexes II and III, in which PKI transitions from the A to P site, while eEF2 occupies the A site partially. By comparison, the similarly mid-rotated (4°) 80S•TSV IRES initiation complex, in the absence of eEF2 (Koh et al., 2014), adopts a mid-swiveled position (~10°) (Figure 2c). These observations suggest that eEF2 is necessary for inducing or stabilizing the large head swivel of the 40S subunit characteristic for IRES translocation intermediates.

IRES rearrangements

In each structure, the TSV IRES adopts a distinct conformation in the intersubunit space of the ribosome (Figures 3 and 4). The IRES (nt 6758–6952) consists of two globular parts (Figure 3a): the 5’-region (domains I and II, nt 6758–6888) and the PKI domain (domain III, nt 6889–6952). We collectively term domains I and II the 5’ domain. The PKI domain comprises PKI and stem loop 3 (SL3), which stacks on top of the stem of PKI (Koh et al., 2014). The ⁶⁹⁵³GCU triplet immediately following the PKI domain is the first codon of the open reading frame. In the eEF2-free 80S•IRES initiation complex (INIT) (Koh et al., 2014), the bulk of the 5’-domain (nt. 6758–6888) binds near the E site, contacting the ribosome mostly by means of three protruding structural elements: the L1.1 region and stem loops 4 and 5 (SL4 and SL5). In Structures I to IV, these contacts remain as in the initiation complex (Figure 1a). Specifically, the L1.1 region interacts with the L1 stalk of the large subunit, while SL4 and SL5 bind at the side of the 40S head and interact with proteins uS7, uS11 and eS25 (Figure 3—figure supplement 2 and Figure 3—figure supplement 3; ribosomal proteins are termed according to Ban et al., 2014). In Structures I-IV, the minor groove of SL4 (at nt 6840–6846) binds next to an α-helix of uS7, which is rich in positively charged residues (K212, K213, R219 and K222). The tip of SL4 binds in the vicinity of R157 in the β-hairpin of uS7 and of Y58 in uS11. The minor groove of SL5 (at nt 6862–6868) contacts the positively charged region of eS25 (R49, R58 and R68) (Figure 3—figure supplement 4). In Structure V, however, the density for SL5 is missing suggesting that SL5 is mobile, while weak SL4 density suggests that SL4 is shifted along the surface of uS7, ~20 Å away from its initial position (Figure 3—figure supplement 2c). The L1.1 region remains in contact with the L1 stalk (Figure 3—figure supplement 3).

Figure 3 with 7 supplements see all

Download asset Open asset

The shape of the IRES changes considerably from the initiation state to Structures I through V, from an extended to compact to extended conformation (Figure 4; see also Figure 3—figure supplement 2a). Because in Structures I to IV the PKI domain shifts toward the P site, while the 5’ remains unchanged near the E site, the distance between the domains shortens (Figure 4). In the 80S•IRES initiation state (Koh et al., 2014), the A-site-bound PKI is separated from SL4 by almost 50 Å (Figure 4). In Structures I and II, the PKI is partially retracted from the A site and the distance from SL4 shortens to ~35 Å. As PKI moves toward the P site in Structures III and IV, the PKI domain approaches to within ~25 Å of SL4. Because the 5’-domain in the following structure (V) moves by ~20 Å along the 40S head, the IRES returns to an extended conformation (~45 Å) that is similar to that in the 80S•IRES initiation complex.

Figure 4

Download asset Open asset

Rearrangements of the IRES involve restructuring of several interactions with the ribosome. In Structure I, SL3 of the PKI domain is positioned between the A-site finger (nt 1008–1043 of 25S rRNA) and the P site of the 60S subunit, comprising helix 84 of 25S rRNA (nt. 2668–2687) and protein uL5 (Figure 3—figure supplement 6). This position of SL3 is ~25 Å away from that in the 80S•IRES initiation state (Koh et al., 2014), in which PKI and SL3 closely mimic the ASL and elbow of the A-site tRNA, respectively (Koh et al., 2014). As such, the transition from the initiation state to Structure I involves repositioning of SL3 around the A-site finger, resembling the transition between the pre-translocation A/P and A/P* tRNA (Brilot et al., 2013). The second set of major structural changes involves interaction of the P site region of the large subunit with the hinge point of the IRES bending between the 5´ domain and the PKI domain (nt. 6886–6890). In the highly bent Structures III and IV, the hinge region interacts with the universally conserved uL5 and the C-terminal tail of eL42 (Figure 3—figure supplement 7). However, in the extended conformations, these parts of the IRES and the 60S subunit are separated by more than 10 Å, suggesting that an interaction between them stabilizes the bent conformations but not the extended ones. Another local rearrangement concerns loop 3, also known as the variable loop region (Ruehle et al., 2015; Ren et al., 2014; Au and Jan, 2012), which connects the ASL- and mRNA-like parts of PKI. This loop is poorly resolved in Structures I through IV, suggesting conformational flexibility in agreement with structural studies of the isolated PKI (Costantino et al., 2008; Zhu et al., 2011) and biochemical studies of unbound IRESs (Jan and Sarnow, 2002; Pfingsten et al., 2010). In Structure V, loop 3 is bound in the 40S E site and the backbone of loop 3 near the codon-like part of PKI (at nt. 6945–6946) interacts with R148 and R157 in β-hairpin of uS7. The interaction of loop 3 backbone with uS7 resembles that of the anticodon-stem loop of E-site tRNA in the post-translocation 80S•2tRNA•mRNA structure (Figure 3—figure supplement 5) (Svidritskiy et al., 2014). Ordering of loop 3 suggests that this flexible region contributes to the stabilization of the PKI domain in the post-translocation state. This interpretation is consistent with the recent observation that alterations in loop 3 of the CrPV IRES result in decreased efficiency of translocation (Ruehle et al., 2015).

eEF2 structures

Elongation factor eEF2 in all five structures is bound with GDP and sordarin (Figure 5). The elongation factor consists of three dynamic superdomains (Jorgensen et al., 2003): an N-terminal globular superdomain formed by the G (GTPase) domain (domain I) and domain II; a linker domain III; and a C-terminal superdomain comprising domains IV and V (Figure 5a). Domain IV extends from the main body and is critical for translocation catalyzed by eEF2 or EF-G. ADP-ribosylation of eEF2 at the tip of domain IV (Davydova and Ovchinnikov, 1990; Nygard and Nilsson, 1990) or deletion of domain IV from EF-G (Martemyanov and Gudkov, 1999; Rodnina et al., 1997) abrogate translocation. In post-translocation-like 80S•tRNA•eEF2 complexes, domain IV binds in the 40S A site, suggesting direct involvement of domain IV in translocation of tRNA from the A to P site (Spahn et al., 2004a; Taylor et al., 2007). GDP in our structures is bound in the GTPase center (Figures 5d, e and f) and sordarin is sandwiched between the β-platforms of domains III and V (Figures 5g and h), as in the structure of free eEF2•sordarin complex (Jorgensen et al., 2003).

Figure 5 with 2 supplements see all

Download asset Open asset

The global conformations of eEF2 (Figure 5a) are similar in these structures (all-atom RMSD ≤ 2 Å), but the positions of eEF2 relative to the 40S subunit differ substantially as a result of 40S subunit rotation (Figure 2—source data 1). From Structure I to V, eEF2 is rigidly attached to the GTPase-associated center of the 60S subunit. The GTPase-associated center comprises the P stalk (L11 and L7/L12 stalk in bacteria) and the sarcin-ricin loop (SRL, nt 3012–3042). The tips of 25S rRNA helices 43 and 44 of the P stalk (nucleotides G1242 and A1270, respectively) stack on V754 and Y744 of domain V. An αββ motif of the eukaryote-specific protein P0 (aa 126–154) packs in the crevice between the long α-helix D (aa 172–188) of the GTPase domain and the β-sheet region (aa 246–263) of the GTPase domain insert (or G’ insert) of eEF2 (secondary-structure nomenclatures for eEF2 and EF-G (Czworkowski et al., 1994) are the same). Although the P/L11 stalk is known to be dynamic (Korostelev et al., 2008; Taylor et al., 2012), its position remains unchanged from Structure I to V: all-atom root-mean-square differences for the 25S rRNA of the P stalk (nt 1223–1286) are within 2.5 Å. However, with respect to its position in the 80S•IRES complex in the absence of eEF2 and in the 80S•2tRNA•mRNA complex, the P stalk is shifted by ~13 Å toward the A site (Figure 2d). The sarcin-ricin loop interacts with the GTP-binding site of eEF2 (Figures 5d and f). While the overall mode of this interaction is similar to that seen in 70S•EF-G crystal structures (Chen et al., 2013b; Gao et al., 2009; Pulk and Cate, 2013; Tourigny et al., 2013; Zhou et al., 2013; 2014), there is an important local difference between Structure I and Structures II-V in switch loop I, as discussed below.

There are two modest but noticeable domain rearrangements between Structures I and V. Unlike in free eEF2, which can sample large movements of at least 50 Å of the C-terminal superdomain relative to the N-terminal superdomain (Figure 5c) (Jorgensen et al., 2003), eEF2 undergoes moderate repositioning of domain IV (~3 Å; Figure 5a) and domain III (~5 Å; Figure 6d). This limited flexibility of the ribosome-bound eEF2 is likely the result of simultaneous fixation of eEF2 superdomains, via domains I and V, by the GTPase-associated center of the large subunit. Domain IV of eEF2 binds at the 40S A site in Structures I to V but the mode of interaction differs in each complex (Figure 6). Because eEF2 is rigidly attached to the 60S subunit and does not undergo large inter-subunit rearrangements, gradual entry of domain IV into the A site between Structures I and V is due to 40S subunit rotation and head swivel. eEF2 settles into the A site from Structure I to V, as the tip of domain IV shifts by ~10 Å relative to the body and by ~20 Å relative to the swiveling head. Modest intra-eEF2 shifts of domain IV between Structures I to V outline a stochastic trajectory (Figure 5a), consistent with local adjustments of the domain in the A site. At the central region of eEF2, domains II and III contact the 40S body (mainly at nucleotides 48–52 and 429–432 of 18S rRNA helix 5 and uS12). From Structure I to V, these central domains migrate by ~10 Å along the 40S surface (Figure 6c). Comparison of eEF2 conformations reveals that in Structure V, domain III is displaced as a result of interaction with uS12, as discussed below.

Figure 6 with 1 supplement see all

Download asset Open asset

In summary, between Structures I and V, a step-wise translocation of PKI by ~15 Å from the A to P site - within the 40S subunit – occurs simultaneously with the ~11 Å side-way entry of domain IV into the A site coupled with ~3 to 5 Å inter-domain rearrangements in eEF2. These shifts occur during the reverse rotation of the 40S body coupled with the forward-then-reverse head swivel. To elucidate the detailed structural mechanism of IRES translocation and the roles of eEF2 and ribosome rearrangements, we describe in the following sections the interactions of PKI and eEF2 with the ribosomal A and P sites in Structures I through V (Figure 2g; see also Figure 1—figure supplement 1).

Structure I represents a pre-translocation IRES and initial entry of eEF2 in a GTP-like state

In the fully rotated Structure I, PKI is shifted toward the P site by ~3 Å relative to its position in the initiation complex but maintains interactions with the partially swiveled head. At the head, C1274 of the 18S rRNA (C1054 in E. coli) base pairs with the first nucleotide of the ORF immediately downstream of PKI. The C1274:G6953 base pair provides a stacking platform for the codon-anticodon–like helix of PKI. We therefore define C1274 as the foundation of the 'head A site'. Accordingly, we use U1191 (G966 in E. coli) and C1637 (C1400 in E. coli) as the reference points of the 'head P site' and 'body P site' (Figure 2g), respectively, because these nucleotides form a stacking foundation for the fully translocated mRNA-tRNA helix in tRNA-bound structures (Korostelev et al., 2006; Selmer et al., 2006; Svidritskiy et al., 2014) and in our post-translocation Structure V discussed below.

The interaction of PKI with the 40S body is substantially rearranged relative to that in the initiation state. In the latter, PKI is stabilized by interactions with the universally conserved decoding-center nucleotides G577, A1755 and A1756 ('body A site'), as in the A-site tRNA bound complexes (Koh et al., 2014). In Structure I, PKI does not contact these nucleotides (Figures 2g and 7).

Figure 7

Download asset Open asset

The position of eEF2 on the 40S subunit of Structure I is markedly distinct from those in Structures II to V. The translocase interacts with the 40S body but does not contact the head (Figures 5b and 6a; Figure 5—figure supplement 1). Domain IV is partially engaged with the body A site. The tip of domain IV is wedged between PKI and decoding-center nucleotides A1755 and A1756, which are bulged out of h44. This tip contains the histidine-diphthamide triad (H583, H694 and Diph699), which interacts with the codon-anticodon-like helix of PKI and A1756 (Figure 7). Histidines 583 and 694 interact with the phosphate backbone of the anticodon-like strand (at G6907 and C6908). Diphthamide is a unique posttranslational modification conserved in archaeal and eukaryotic EF2 (at residue 699 in S. cerevisiae) and involves addition of a ~7-Å long 3-carboxyamido-3-(trimethylamino)-propyl moiety to the histidine imidazole ring at CE1. The trimethylamino end of Diph699 packs over A1756 (Figure 7). The opposite surface of the tail is oriented toward the minor-groove side of the second base pair of the codon-anticodon helix (G6906:C6951). Thus, in comparison with the initiation state, the histidine-diphthamide tip of eEF2 replaces the codon-anticodon–like helix of PKI. The splitting of the interaction of A1755-A1756 and PKI is achieved by providing the histidine-diphthamine tip as a binding partner for both A1756 and the minor groove of the codon-anticodon helix (Figure 7).

Unlike in Structures II to V, the conformation of the eEF2 GTPase center in Structure I resembles that of a GTP-bound translocase (Figure 5e). In translational GTPases, switch loops I and II are involved in the GTPase activity (reviewed in Voorhees and Ramakrishnan, (2013)). Switch loop II (aa 105–110), which carries the catalytic H108 (H92 in E. coli EF-G; (Cunha et al., 2013; Holtkamp et al., 2014; Koripella et al., 2015; Salsi et al., 2014) is well resolved in all five structures. The histidine resides next to the backbone of G3028 of the sarcin-ricin loop and near the diphosphate of GDP (Figure 5e). By contrast, switch loop I (aa 50–70 in S. cerevisiae eEF2) is resolved only in Structure I (Figure 5—figure supplement 2). The N-terminal part of the loop (aa 50–60) is sandwiched between the tip of helix 14 (⁴¹⁵CAAA⁴¹⁸) of the 18S rRNA of the 40S subunit and helix A (aa 32–42) of eEF2 (Figure 5d). Bulged A416 interacts with the switch loop in the vicinity of D53. Next to GDP, the C-terminal part of the switch loop (aa 61–67) adopts a helical fold. As such, the conformations of SWI and the GTPase center in general are similar to those observed in ribosome-bound EF-Tu (Voorhees et al., 2010) and EF-G (Pulk and Cate, 2013; Tourigny et al., 2013; Zhou et al., 2013) in the presence of GTP analogs.

1. 1. Priyanka D Abeyrathne
   2. Cha San Koh
   3. Timothy Grant
   4. Nikolaus Grigorieff
   5. Andrei A Korostelev

   (2016) Saccharomyces cerevisiae 80S ribosome bound with elongation factor eEF2-GDP-sordarin and Taura Syndrome Virus IRES, Structure I (fully rotated 40S subunit)

   Publicly available at the RCSB Protein Data Bank (accession no. 5JUO).

   http://www.rcsb.org/pdb/explore/explore.do?structureId=5JUO
2. 1. Priyanka D Abeyrathne
   2. Cha San Koh
   3. Timothy Grant
   4. Nikolaus Grigorieff
   5. Andrei A Korostelev

   (2016) Saccharomyces cerevisiae 80S ribosome bound to elongation factor eEF2-GDP-sordarin and Taura Syndrome Virus IRES, Structure I

   Publicly available at the Electron Microscopy Data Bank (accession no. EMD-6643).

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-6643
3. 1. Priyanka D Abeyrathne
   2. Cha San Koh
   3. Timothy Grant
   4. Nikolaus Grigorieff
   5. Andrei A Korostelev

   (2016) Saccharomyces cerevisiae 80S ribosome bound to TSV IRES and eEF2 translation initiation complex, Structure I, Map 2

   Publicly available at the Electron Microscopy Data Bank (accession no. EMD-6648).

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-6648
4. 1. Priyanka D Abeyrathne
   2. Cha San Koh
   3. Timothy Grant
   4. Nikolaus Grigorieff
   5. Andrei A Korostelev

   (2016) Saccharomyces cerevisiae 80S ribosome bound to elongation factor eEF2-GDP-sordarin and Taura Syndrome Virus IRES, Structure II (mid-rotated 40S subunit)

   Publicly available at the RCSB Protein Data Bank (accession no. 5JUP).

   http://www.rcsb.org/pdb/explore/explore.do?structureId=5JUP
5. 1. Priyanka D Abeyrathne
   2. Cha San Koh
   3. Timothy Grant
   4. Nikolaus Grigorieff
   5. Andrei A Korostelev

   (2016) Saccharomyces cerevisiae 80S ribosome bound to elongation factor eEF2-GDP-sordarin and Taura Syndrome Virus IRES, Structure II (mid-rotated 40S subunit)

   Publicly available at the Electron Microscopy Data Bank (accession no. EMD-6644).

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-6644
6. 1. Priyanka D Abeyrathne
   2. Cha San Koh
   3. Timothy Grant
   4. Nikolaus Grigorieff
   5. Andrei A Korostelev

   (2016) Saccharomyces cerevisiae 80S ribosome bound to TSV IRES and eEF2 translation initiation complex, Structure II, Map 2

   Publicly available at the Electron Microscopy Data Bank (accession no. EMD-6649).

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-6649
7. 1. Priyanka D Abeyrathne
   2. Cha San Koh
   3. Timothy Grant
   4. Nikolaus Grigorieff
   5. Andrei A Korostelev

   (2016) Saccharomyces cerevisiae 80S ribosome bound with elongation factor eEF2-GDP-sordarin and Taura Syndrome Virus IRES, Structure III (mid-rotated 40S subunit)

   Publicly available at the RCSB Protein Data Bank (accession no. 5JUS).

   http://www.rcsb.org/pdb/explore/explore.do?structureId=5JUS
8. 1. Priyanka D Abeyrathne
   2. Cha San Koh
   3. Timothy Grant
   4. Nikolaus Grigorieff
   5. Andrei A Korostelev

   (2016) Saccharomyces cerevisiae 80S ribosome bound to elongation factor eEF2-GDP-sordarin and Taura Syndrome Virus IRES, Structure III (mid-rotated 40S subunit)

   Publicly available at the Electron Microscopy Data Bank (accession no. EMD-6645).

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-6645
9. 1. Priyanka D Abeyrathne
   2. Cha San Koh
   3. Timothy Grant
   4. Nikolaus Grigorieff
   5. Andrei A Korostelev

   (2016) Saccharomyces cerevisiae 80S ribosome bound to TSV IRES and eEF2 translation initiation complex, Structure III, Map 2

   Publicly available at the Electron Microscopy Data Bank (accession no. EMD-6650).

   https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-6650
10. 1. Priyanka D Abeyrathne
    2. Cha San Koh
    3. Timothy Grant
    4. Nikolaus Grigorieff
    5. Andrei A Korostelev

    (2016) Saccharomyces cerevisiae 80S ribosome bound to TSV IRES and eEF2 translation initiation complex, Structure III, Map 3

    Publicly available at the Electron Microscopy Data Bank (accession no. EMD-6651).

    https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-6651
11. 1. Priyanka D Abeyrathne
    2. Cha San Koh
    3. Timothy Grant
    4. Nikolaus Grigorieff
    5. Andrei A Korostelev

    (2016) Saccharomyces cerevisiae 80S ribosome bound with elongation factor eEF2-GDP-sordarin and Taura Syndrome Virus IRES, structure IV (almost non-rotated 40S subunit)

    Publicly available at the RCSB Protein Data Bank (accession no. 5JUT).

    http://www.rcsb.org/pdb/explore/explore.do?structureId=5JUT
12. 1. Priyanka D Abeyrathne
    2. Cha San Koh
    3. Timothy Grant
    4. Nikolaus Grigorieff
    5. Andrei A Korostelev

    (2016) Saccharomyces cerevisiae 80S ribosome bound to elongation factor eEF2-GDP-sordarin and Taura Syndrome Virus IRES, Structure IV (almost non-rotated 40S subunit)

    Publicly available at the Electron Microscopy Data Bank (accession no. EMD-6646).

    https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-6646
13. 1. Priyanka D Abeyrathne
    2. Cha San Koh
    3. Timothy Grant
    4. Nikolaus Grigorieff
    5. Andrei A Korostelev

    (2016) Saccharomyces cerevisiae 80S ribosome bound to TSV IRES and eEF2 translation initiation complex, Structure IV, Map 2

    Publicly available at the Electron Microscopy Data Bank (accession no. EMD-6652).

    https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-6652
14. 1. Priyanka D Abeyrathne
    2. Cha San Koh
    3. Timothy Grant
    4. Nikolaus Grigorieff
    5. Andrei A Korostelev

    (2016) Saccharomyces cerevisiae 80S ribosome bound with elongation factor eEF2-GDP-sordarin and Taura Syndrome Virus IRES, Structure V (least rotated 40S subunit)

    Publicly available at the RCSB Protein Data Bank (accession no. 5JUU).

    http://www.rcsb.org/pdb/explore/explore.do?structureId=5JUU
15. 1. Priyanka D Abeyrathne
    2. Cha San Koh
    3. Timothy Grant
    4. Nikolaus Grigorieff
    5. Andrei A Korostelev

    (2016) Saccharomyces cerevisiae 80S ribosome bound to elongation factor eEF2-GDP-sordarin and Taura Syndrome Virus IRES, Structure V (least rotated 40S subunit)

    Publicly available at the Electron Microscopy Data Bank (accession no. EMD-6647).

    https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-6647
16. 1. Priyanka D Abeyrathne
    2. Cha San Koh
    3. Timothy Grant
    4. Nikolaus Grigorieff
    5. Andrei A Korostelev

    (2016) Saccharomyces cerevisiae 80S ribosome bound to TSV IRES and eEF2 translation initiation complex, Structure V, Map 2

    Publicly available at the Electron Microscopy Data Bank (accession no. EMD-6653).

    https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-6653

1. 1. AEvarsson A
   2. Brazhnikov E
   3. Garber M
   4. Zheltonosova J
   5. Chirgadze Y
   6. al-Karadaghi S
   7. Svensson LA
   8. Liljas A

   (1994)

   Three-dimensional structure of the ribosomal translocase: elongation factor G from Thermus thermophilus

   The EMBO Journal 13:3669–3677.

   • Google Scholar
2. 1. Agirrezabala X
   2. Lei J
   3. Brunelle JL
   4. Ortiz-Meoz RF
   5. Green R
   6. Frank J

   (2008) Visualization of the hybrid state of trna binding promoted by spontaneous ratcheting of the ribosome

   Molecular Cell 32:190–197.

   https://doi.org/10.1016/j.molcel.2008.10.001

   • Google Scholar
3. 1. Agirrezabala X
   2. Valle M

   (2015) Structural insights into trna dynamics on the ribosome

   International Journal of Molecular Sciences 16:9866–9895.

   https://doi.org/10.3390/ijms16059866

   • Google Scholar
4. 1. Au HH
   2. Jan E

   (2012) Insights into factorless translational initiation by the trna-like pseudoknot domain of a viral IRES

   PloS One 7:e51477.

   https://doi.org/10.1371/journal.pone.0051477

   • Google Scholar
5. 1. Bai XC
   2. Fernandez IS
   3. McMullan G
   4. Scheres SH

   (2013) Ribosome structures to near-atomic resolution from thirty thousand cryo-em particles

   eLife 2:e00461.

   https://doi.org/10.7554/eLife.00461

   • Google Scholar
6. 1. Ban N
   2. Beckmann R
   3. Cate JH
   4. Dinman JD
   5. Dragon F
   6. Ellis SR
   7. Lafontaine DL
   8. Lindahl L
   9. Liljas A
   10. Lipton JM
   11. McAlear MA
   12. Moore PB
   13. Noller HF
   14. Ortega J
   15. Panse VG
   16. Ramakrishnan V
   17. Spahn CM
   18. Steitz TA
   19. Tchorzewski M
   20. Tollervey D
   21. Warren AJ
   22. Williamson JR
   23. Wilson D
   24. Yonath A
   25. Yusupov M

   (2014) A new system for naming ribosomal proteins

   Current Opinion in Structural Biology 24:165–169.

   https://doi.org/10.1016/j.sbi.2014.01.002

   • Google Scholar
7. 1. Ben-Shem A
   2. Garreau de Loubresse N
   3. Melnikov S
   4. Jenner L
   5. Yusupova G
   6. Yusupov M

   (2011) The structure of the eukaryotic ribosome at 3.0 A resolution

   Science 334:1524–1529.

   https://doi.org/10.1126/science.1212642

   • Google Scholar
8. 1. Bermek E

   (1976)

   Interactions of adenosine diphosphate-ribosylated elongation factor 2 with ribosomes

   The Journal of Biological Chemistry 251:6544–6549.

   • Google Scholar
9. 1. Blanchard SC
   2. Kim HD
   3. Gonzalez RL
   4. Puglisi JD
   5. Chu S

   (2004) tRNA dynamics on the ribosome during translation

   Proceedings of the National Academy of Sciences of the United States of America 101:12893–12898.

   https://doi.org/10.1073/pnas.0403884101

   • Google Scholar
10. 1. Bretscher MS

    (1968) Translocation in protein synthesis: A hybrid structure model

    Nature 218:675–677.

    https://doi.org/10.1038/218675a0

    • Google Scholar
11. 1. Brilot AF
    2. Korostelev AA
    3. Ermolenko DN
    4. Grigorieff N

    (2013) Structure of the ribosome with elongation factor G trapped in the pretranslocation state

    Proceedings of the National Academy of Sciences of the United States of America 110:.

    https://doi.org/10.1073/pnas.1311423110

    • Google Scholar
12. 1. Budkevich T
    2. Giesebrecht J
    3. Altman RB
    4. Munro JB
    5. Mielke T
    6. Nierhaus KH
    7. Blanchard SC
    8. Spahn CM

    (2011) Structure and dynamics of the mammalian ribosomal pretranslocation complex

    Molecular Cell 44:214–224.

    https://doi.org/10.1016/j.molcel.2011.07.040

    • Google Scholar
13. 1. Cardone G
    2. Heymann JB
    3. Steven AC

    (2013) One number does not fit all: Mapping local variations in resolution in cryo-em reconstructions

    Journal of Structural Biology 184:226–236.

    https://doi.org/10.1016/j.jsb.2013.08.002

    • Google Scholar
14. 1. Cevallos RC
    2. Sarnow P

    (2005) Factor-independent assembly of elongation-competent ribosomes by an internal ribosome entry site located in an RNA virus that infects penaeid shrimp

    Journal of Virology 79:677–683.

    https://doi.org/10.1128/JVI.79.2.677-683.2005

    • Google Scholar
15. 1. Chapman MS

    (1995) Restrained real-space macromolecular atomic refinement using a new resolution-dependent electron-density function

    Acta Crystallographica Section a Foundations of Crystallography 51:69–80.

    https://doi.org/10.1107/S0108767394007130

    • Google Scholar
16. 1. Chen B
    2. Kaledhonkar S
    3. Sun M
    4. Shen B
    5. Lu Z
    6. Barnard D
    7. Lu TM
    8. Gonzalez RL
    9. Frank J

    (2015) Structural dynamics of ribosome subunit association studied by mixing-spraying time-resolved cryogenic electron microscopy

    Structure 23:1097–1105.

    https://doi.org/10.1016/j.str.2015.04.007

    • Google Scholar
17. 1. Chen J
    2. Petrov A
    3. Tsai A
    4. O'Leary SE
    5. Puglisi JD

    (2013a) Coordinated conformational and compositional dynamics drive ribosome translocation

    Nature Structural & Molecular Biology 20:718–727.

    https://doi.org/10.1038/nsmb.2567

    • Google Scholar
18. 1. Chen Y
    2. Feng S
    3. Kumar V
    4. Ero R
    5. Gao Y-G

    (2013b) Structure of ef-g–ribosome complex in a pretranslocation state

    Nature Structural & Molecular Biology 20:1077–1084.

    https://doi.org/10.1038/nsmb.2645

    • Google Scholar
19. 1. Cheng Y
    2. Grigorieff N
    3. Penczek PA
    4. Walz T

    (2015) A primer to single-particle cryo-electron microscopy

    Cell 161:438–449.

    https://doi.org/10.1016/j.cell.2015.03.050

    • Google Scholar
20. 1. Collier RJ

    (2001) Understanding the mode of action of diphtheria toxin: A perspective on progress during the 20th century

    Toxicon : Official Journal of the International Society on Toxinology 39:1793–1803.

    https://doi.org/10.1016/S0041-0101(01)00165-9

    • Google Scholar
21. 1. Colussi TM
    2. Costantino DA
    3. Zhu J
    4. Donohue JP
    5. Korostelev AA
    6. Jaafar ZA
    7. Plank T-DM
    8. Noller HF
    9. Kieft JS

    (2015) Initiation of translation in bacteria by a structured eukaryotic IRES RNA

    Nature 519:110–113.

    https://doi.org/10.1038/nature14219

    • Google Scholar
22. 1. Cornish PV
    2. Ermolenko DN
    3. Noller HF
    4. Ha T

    (2008) Spontaneous intersubunit rotation in single ribosomes

    Molecular Cell 30:578–588.

    https://doi.org/10.1016/j.molcel.2008.05.004

    • Google Scholar
23. 1. Costantino DA
    2. Pfingsten JS
    3. Rambo RP
    4. Kieft JS

    (2008) tRNA-mrna mimicry drives translation initiation from a viral IRES

    Nature Structural & Molecular Biology 15:57–64.

    https://doi.org/10.1038/nsmb1351

    • Google Scholar
24. 1. Cukras AR
    2. Southworth DR
    3. Brunelle JL
    4. Culver GM
    5. Green R

    (2003) Ribosomal proteins S12 and S13 function as control elements for translocation of the mrna:trna complex

    Molecular Cell 12:321–328.

    https://doi.org/10.1016/S1097-2765(03)00275-2

    • Google Scholar
25. 1. Cunha CE
    2. Belardinelli R
    3. Peske F
    4. Holtkamp W
    5. Wintermeyer W
    6. Rodnina MV

    (2013) Dual use of GTP hydrolysis by elongation factor G on the ribosome

    Translation 1:e24315.

    https://doi.org/10.4161/trla.24315

    • Google Scholar
26. 1. Czworkowski J
    2. Wang J
    3. Steitz TA
    4. Moore PB

    (1994)

    The crystal structure of elongation factor G complexed with GDP, at 2.7 A resolution

    The EMBO Journal 13:3661–3668.

    • Google Scholar
27. 1. Davydova EK
    2. Ovchinnikov LP

    (1990) ADP-ribosylated elongation factor 2 (adp-ribosyl-ef-2) is unable to promote translocation within the ribosome

    FEBS Letters 261:350–352.

    https://doi.org/10.1016/0014-5793(90)80589-B

    • Google Scholar
28. 1. Deforges J
    2. Locker N
    3. Sargueil B

    (2015) mRNAs that specifically interact with eukaryotic ribosomal subunits

    Biochimie 114:48–57.

    https://doi.org/10.1016/j.biochi.2014.12.008

    • Google Scholar
29. Software

    1. DeLano WL

    (2002) The Pymol Molecular Graphics System

    DeLano Scientific, Palo Alto, CA.

    http://www.pymol.org
30. 1. Demeshkina N
    2. Jenner L
    3. Westhof E
    4. Yusupov M
    5. Yusupova G

    (2012) A new understanding of the decoding principle on the ribosome

    Nature 484:256–259.

    https://doi.org/10.1038/nature10913

    • Google Scholar
31. 1. Domínguez JM
    2. Gómez-Lorenzo MG
    3. Martín JJ

    (1999) Sordarin inhibits fungal protein synthesis by blocking translocation differently to fusidic acid

    The Journal of Biological Chemistry 274:22423–22427.

    https://doi.org/10.1074/jbc.274.32.22423

    • Google Scholar
32. 1. Dunkle JA
    2. Cate JH

    (2010) Ribosome structure and dynamics during translocation and termination

    Annual Review of Biophysics 39:227–244.

    https://doi.org/10.1146/annurev.biophys.37.032807.125954

    • Google Scholar
33. 1. Emsley P
    2. Cowtan K

    (2004) Coot: Model-building tools for molecular graphics

    Acta Crystallographica. Section D, Biological Crystallography 60:2126–2132.

    https://doi.org/10.1107/S0907444904019158

    • Google Scholar
34. 1. Ermolenko DN
    2. Cornish PV
    3. Ha T
    4. Noller HF

    (2013) Antibiotics that bind to the A site of the large ribosomal subunit can induce mrna translocation

    RNA 19:158–166.

    https://doi.org/10.1261/rna.035964.112

    • Google Scholar
35. 1. Ermolenko DN
    2. Majumdar ZK
    3. Hickerson RP
    4. Spiegel PC
    5. Clegg RM
    6. Noller HF

    (2007) Observation of intersubunit movement of the ribosome in solution using FRET

    Journal of Molecular Biology 370:530–540.

    https://doi.org/10.1016/j.jmb.2007.04.042

    • Google Scholar
36. 1. Ermolenko DN
    2. Noller HF

    (2011) mRNA translocation occurs during the second step of ribosomal intersubunit rotation

    Nature Structural & Molecular Biology 18:457–462.

    https://doi.org/10.1038/nsmb.2011

    • Google Scholar
37. 1. Fei J
    2. Kosuri P
    3. MacDougall DD
    4. Gonzalez RL

    (2008) Coupling of ribosomal L1 stalk and trna dynamics during translation elongation

    Molecular Cell 30:348–359.

    https://doi.org/10.1016/j.molcel.2008.03.012

    • Google Scholar
38. 1. Fernández IS
    2. Bai XC
    3. Murshudov G
    4. Scheres SH
    5. Ramakrishnan V

    (2014) Initiation of translation by cricket paralysis virus IRES requires its translocation in the ribosome

    Cell 157:823–831.

    https://doi.org/10.1016/j.cell.2014.04.015

    • Google Scholar
39. 1. Fischer N
    2. Konevega AL
    3. Wintermeyer W
    4. Rodnina MV
    5. Stark H

    (2010) Ribosome dynamics and trna movement by time-resolved electron cryomicroscopy

    Nature 466:329–333.

    https://doi.org/10.1038/nature09206

    • Google Scholar
40. 1. Frank J
    2. Agrawal RK

    (2000) A ratchet-like inter-subunit reorganization of the ribosome during translocation

    Nature 406:318–322.

    https://doi.org/10.1038/35018597

    • Google Scholar
41. 1. Gao Y-G
    2. Selmer M
    3. Dunham CM
    4. Weixlbaumer A
    5. Kelley AC
    6. Ramakrishnan V

    (2009) The structure of the ribosome with elongation factor G trapped in the posttranslocational state

    Science 326:694–699.

    https://doi.org/10.1126/science.1179709

    • Google Scholar
42. 1. Gavrilova LP
    2. Kostiashkina OE
    3. Koteliansky VE
    4. Rutkevitch NM
    5. Spirin AS

    (1976) Factor-free ("non-enzymic") and factor-dependent systems of translation of polyuridylic acid by escherichia coli ribosomes

    Journal of Molecular Biology 101:537–552.

    https://doi.org/10.1016/0022-2836(76)90243-6

    • Google Scholar
43. 1. Gavrilova LP
    2. Spirin AS

    (1972)

    Mechanism of translocation in ribosomes. II. Activation of spontaneous (nonenzymic) translocation in ribosomes of Escherichia coli by p-chloromercuribenzoate

    Molecular Biology 6:248–254.

    • Google Scholar
44. 1. Givaty O
    2. Levy Y

    (2009) Protein sliding along DNA: dynamics and structural characterization

    Journal of Molecular Biology 385:1087–1184.

    https://doi.org/10.1016/j.jmb.2008.11.016

    • Google Scholar
45. 1. Gonen T
    2. Cheng Y
    3. Sliz P
    4. Hiroaki Y
    5. Fujiyoshi Y
    6. Harrison SC
    7. Walz T

    (2005) Lipid–protein interactions in double-layered two-dimensional AQP0 crystals

    Nature 438:633–638.

    https://doi.org/10.1038/nature04321

    • Google Scholar
46. 1. Gorman J
    2. Plys AJ
    3. Visnapuu ML
    4. Alani E
    5. Greene EC

    (2010) Visualizing one-dimensional diffusion of eukaryotic DNA repair factors along a chromatin lattice

    Nature Structural & Molecular Biology 17:932–940.

    https://doi.org/10.1038/nsmb.1858

    • Google Scholar
47. 1. Grant T
    2. Grigorieff N

    (2015a) Automatic estimation and correction of anisotropic magnification distortion in electron microscopes

    Journal of Structural Biology 192:204–208.

    https://doi.org/10.1016/j.jsb.2015.08.006

    • Google Scholar
48. 1. Grant T
    2. Grigorieff N

    (2015b) Measuring the optimal exposure for single particle cryo-EM using a 2.6 Å reconstruction of rotavirus VP6

    eLife 4:e06980.

    https://doi.org/10.7554/eLife.06980

    • Google Scholar
49. 1. Guo Z
    2. Noller HF

    (2012) Rotation of the head of the 30S ribosomal subunit during mrna translocation

    Proceedings of the National Academy of Sciences of the United States of America 109:20391–20394.

    https://doi.org/10.1073/pnas.1218999109

    • Google Scholar
50. 1. Halford SE

    (2009) An end to 40 years of mistakes in DNA-protein association kinetics?

    Biochemical Society Transactions 37:343–351.

    https://doi.org/10.1042/BST0370343

    • Google Scholar
51. 1. Hatakeyama Y
    2. Shibuya N
    3. Nishiyama T
    4. Nakashima N

    (2004) Structural variant of the intergenic internal ribosome entry site elements in dicistroviruses and computational search for their counterparts

    RNA 10:779–786.

    https://doi.org/10.1261/rna.5208104

    • Google Scholar
52. 1. Holtkamp W
    2. Cunha CE
    3. Peske F
    4. Konevega AL
    5. Wintermeyer W
    6. Rodnina MV

    (2014) GTP hydrolysis by EF-G synchronizes trna movement on small and large ribosomal subunits

    The EMBO Journal 33:1073–1085.

    https://doi.org/10.1002/embj.201387465

    • Google Scholar
53. 1. Horan LH
    2. Noller HF

    (2007) Intersubunit movement is required for ribosomal translocation

    Proceedings of the National Academy of Sciences of the United States of America 104:4881–4885.

    https://doi.org/10.1073/pnas.0700762104

    • Google Scholar
54. 1. Jackson RJ
    2. Hellen CU
    3. Pestova TV

    (2010) The mechanism of eukaryotic translation initiation and principles of its regulation

    Nature Reviews. Molecular Cell Biology 11:113–127.

    https://doi.org/10.1038/nrm2838

    • Google Scholar
55. 1. Jan E
    2. Kinzy TG
    3. Sarnow P

    (2003) Divergent trna-like element supports initiation, elongation, and termination of protein biosynthesis

    Proceedings of the National Academy of Sciences of the United States of America 100:15410–15415.

    https://doi.org/10.1073/pnas.2535183100

    • Google Scholar
56. 1. Jan E
    2. Sarnow P

    (2002) Factorless ribosome assembly on the internal ribosome entry site of cricket paralysis virus

    Journal of Molecular Biology 324:889–902.

    https://doi.org/10.1016/S0022-2836(02)01099-9

    • Google Scholar
57. 1. Jan E
    2. Thompson SR
    3. Wilson JE
    4. Pestova TV
    5. Hellen CU
    6. Sarnow P

    (2001) Initiator met-trna-independent translation mediated by an internal ribosome entry site element in cricket paralysis virus-like insect viruses

    Cold Spring Harbor Symposia on Quantitative Biology 66:285–292.

    https://doi.org/10.1101/sqb.2001.66.285

    • Google Scholar
58. 1. Jenner L
    2. Demeshkina N
    3. Yusupova G
    4. Yusupov M

    (2010) Structural rearrangements of the ribosome at the trna proofreading step

    Nature Structural & Molecular Biology 17:1072–1078.

    https://doi.org/10.1038/nsmb.1880

    • Google Scholar
59. 1. Jørgensen R
    2. Merrill AR
    3. Yates SP
    4. Marquez VE
    5. Schwan AL
    6. Boesen T
    7. Andersen GR

    (2005) Exotoxin a–eef2 complex structure indicates ADP ribosylation by ribosome mimicry

    Nature 436:979–984.

    https://doi.org/10.1038/nature03871

    • Google Scholar
60. 1. Joseph S
    2. Noller HF

    (1998) EF-g-catalyzed translocation of anticodon stem-loop analogs of transfer RNA in the ribosome

    The EMBO Journal 17:3478–3483.

    https://doi.org/10.1093/emboj/17.12.3478

    • Google Scholar
61. 1. Joseph S

    (2003) After the ribosome structure: How does translocation work?

    RNA 9:160–164.

    https://doi.org/10.1261/rna.2163103

    • Google Scholar
62. 1. Julián P
    2. Konevega AL
    3. Scheres SH
    4. Lázaro M
    5. Gil D
    6. Wintermeyer W
    7. Rodnina MV
    8. Valle M

    (2008) Structure of ratcheted ribosomes with trnas in hybrid states

    Proceedings of the National Academy of Sciences of the United States of America 105:16924–16927.

    https://doi.org/10.1073/pnas.0809587105

    • Google Scholar
63. 1. Justice MC
    2. Hsu MJ
    3. Tse B
    4. Ku T
    5. Balkovec J
    6. Schmatz D
    7. Nielsen J

    (1998) Elongation factor 2 as a novel target for selective inhibition of fungal protein synthesis

    The Journal of Biological Chemistry 273:3148–3151.

    https://doi.org/10.1074/jbc.273.6.3148

    • Google Scholar
64. 1. Jørgensen R
    2. Ortiz PA
    3. Carr-Schmid A
    4. Nissen P
    5. Kinzy TG
    6. Andersen GR

    (2003) Two crystal structures demonstrate large conformational changes in the eukaryotic ribosomal translocase

    Nature Structural Biology 10:379–385.

    https://doi.org/10.1038/nsb923

    • Google Scholar
65. 1. Kelley LA
    2. Mezulis S
    3. Yates CM
    4. Wass MN
    5. Sternberg MJ

    (2015) The Phyre2 web portal for protein modeling, prediction and analysis

    Nature Protocols 10:845–903.

    https://doi.org/10.1038/nprot.2015.053

    • Google Scholar
66. 1. Koh CS
    2. Brilot AF
    3. Grigorieff N
    4. Korostelev AA

    (2014) Taura syndrome virus IRES initiates translation by binding its trna-mrna-like structural element in the ribosomal decoding center

    Proceedings of the National Academy of Sciences of the United States of America 111:9139–9144.

    https://doi.org/10.1073/pnas.1406335111

    • Google Scholar
67. 1. Korennykh AV
    2. Piccirilli JA
    3. Correll CC

    (2006) The electrostatic character of the ribosomal surface enables extraordinarily rapid target location by ribotoxins

    Nature Structural & Molecular Biology 13:436–479.

    https://doi.org/10.1038/nsmb1082

    • Google Scholar
68. 1. Koripella RK
    2. Holm M
    3. Dourado D
    4. Mandava CS
    5. Flores S
    6. Sanyal S

    (2015) A conserved histidine in switch-ii of EF-G moderates release of inorganic phosphate

    Scientific Reports 5:.

    https://doi.org/10.1038/srep12970

    • Google Scholar
69. 1. Korostelev A
    2. Bertram R
    3. Chapman MS

    (2002) Simulated-annealing real-space refinement as a tool in model building

    Acta Crystallographica. Section D, Biological Crystallography 58:761–767.

    https://doi.org/10.1107/S0907444902003402

    • Google Scholar
70. 1. Korostelev A
    2. Ermolenko DN
    3. Noller HF

    (2008) Structural dynamics of the ribosome

    Current Opinion in Chemical Biology 12:674–683.

    https://doi.org/10.1016/j.cbpa.2008.08.037

    • Google Scholar
71. 1. Korostelev A
    2. Trakhanov S
    3. Laurberg M
    4. Noller HF

    (2006) Crystal structure of a 70S ribosome-trna complex reveals functional interactions and rearrangements

    Cell 126:1065–1077.

    https://doi.org/10.1016/j.cell.2006.08.032

    • Google Scholar
72. 1. Laurberg M
    2. Asahara H
    3. Korostelev A
    4. Zhu J
    5. Trakhanov S
    6. Noller HF

    (2008) Structural basis for translation termination on the 70S ribosome

    Nature 454:852–857.

    https://doi.org/10.1038/nature07115

    • Google Scholar
73. 1. Lin J
    2. Gagnon MG
    3. Bulkley D
    4. Steitz TA

    (2015) Conformational changes of elongation factor G on the ribosome during tRNA translocation

    Cell 160:219–246.

    https://doi.org/10.1016/j.cell.2014.11.049

    • Google Scholar
74. 1. Ling C
    2. Ermolenko DN

    (2016) Structural insights into ribosome translocation

    Wiley Interdisciplinary Reviews. RNA.

    https://doi.org/10.1002/wrna.1354

    • Google Scholar
75. 1. Liu T
    2. Kaplan A
    3. Alexander L
    4. Yan S
    5. Wen JD
    6. Lancaster L
    7. Wickersham CE
    8. Fredrick K
    9. Fredrik K
    10. Noller H
    11. Tinoco I
    12. Bustamante CJ

    (2014) Direct measurement of the mechanical work during translocation by the ribosome

    eLife 3:e03406.

    https://doi.org/10.7554/eLife.03406

    • Google Scholar
76. 1. Lozano G
    2. Martínez-Salas E

    (2015) Structural insights into viral ires-dependent translation mechanisms

    Current Opinion in Virology 12:113–120.

    https://doi.org/10.1016/j.coviro.2015.04.008

    • Google Scholar
77. 1. Lyumkis D
    2. Brilot AF
    3. Theobald DL
    4. Grigorieff N

    (2013) Likelihood-based classification of cryo-em images using FREALIGN

    Journal of Structural Biology 183:377–388.

    https://doi.org/10.1016/j.jsb.2013.07.005

    • Google Scholar
78. 1. Martemyanov KA
    2. Gudkov AT

    (1999) Domain IV of elongation factor G from Thermus thermophilus is strictly required for translocation

    FEBS Letters 452:155–159.

    https://doi.org/10.1016/S0014-5793(99)00635-3

    • Google Scholar
79. 1. Moazed D
    2. Noller HF

    (1989) Intermediate states in the movement of transfer RNA in the ribosome

    Nature 342:142–148.

    https://doi.org/10.1038/342142a0

    • Google Scholar
80. 1. Moore GE

    (1965)

    Cramming more components onto integrated circuits

    Electronics 86:114–117.

    • Google Scholar
81. 1. Muhs M
    2. Hilal T
    3. Mielke T
    4. Skabkin MA
    5. Sanbonmatsu KY
    6. Pestova TV
    7. Spahn CM

    (2015) Cryo-EM of ribosomal 80S complexes with termination factors reveals the translocated cricket paralysis virus IRES

    Molecular Cell 57:422–432.

    https://doi.org/10.1016/j.molcel.2014.12.016

    • Google Scholar
82. 1. Nishiyama T
    2. Yamamoto H
    3. Shibuya N
    4. Hatakeyama Y
    5. Hachimori A
    6. Uchiumi T
    7. Nakashima N

    (2003) Structural elements in the internal ribosome entry site of plautia stali intestine virus responsible for binding with ribosomes

    Nucleic Acids Research 31:2434–2442.

    https://doi.org/10.1093/nar/gkg336

    • Google Scholar
83. 1. Nygård O
    2. Nilsson L

    (1990)

    Kinetic determination of the effects of ADP-ribosylation on the interaction of eukaryotic elongation factor 2 with ribosomes

    The Journal of Biological Chemistry 265:6030–6034.

    • Google Scholar
84. 1. Ogle JM
    2. Brodersen DE
    3. Clemons WM
    4. Tarry MJ
    5. Carter AP
    6. Ramakrishnan V

    (2001) Recognition of cognate transfer RNA by the 30S ribosomal subunit

    Science 292:897–902.

    https://doi.org/10.1126/science.1060612

    • Google Scholar
85. 1. Pan D
    2. Kirillov SV
    3. Cooperman BS

    (2007) Kinetically competent intermediates in the translocation step of protein synthesis

    Molecular Cell 25:519–529.

    https://doi.org/10.1016/j.molcel.2007.01.014

    • Google Scholar
86. 1. Pestka S

    (1968) Studies on the formation of trensfer ribonucleic acid-ribosome complexes. synthesis

    Proceedings of the National Academy of Sciences of the United States of America 61:726–733.

    https://doi.org/10.1073/pnas.61.2.726

    • Google Scholar
87. 1. Pestova TV
    2. Hellen CU

    (2003) Translation elongation after assembly of ribosomes on the cricket paralysis virus internal ribosomal entry site without initiation factors or initiator trna

    Genes & Development 17:181–186.

    https://doi.org/10.1101/gad.1040803

    • Google Scholar
88. 1. Pettersen EF
    2. Goddard TD
    3. Huang CC
    4. Couch GS
    5. Greenblatt DM
    6. Meng EC
    7. Ferrin TE

    (2004) UCSF chimera--a visualization system for exploratory research and analysis

    Journal of Computational Chemistry 25:1605–1612.

    https://doi.org/10.1002/jcc.20084

    • Google Scholar
89. 1. Pfingsten JS
    2. Castile AE
    3. Kieft JS

    (2010) Mechanistic role of structurally dynamic regions in dicistroviridae IGR iress

    Journal of Molecular Biology 395:205–217.

    https://doi.org/10.1016/j.jmb.2009.10.047

    • Google Scholar
90. 1. Pulk A
    2. Cate JHD

    (2013) Control of ribosomal subunit rotation by elongation factor G

    Science 340:1235970.

    https://doi.org/10.1126/science.1235970

    • Google Scholar
91. 1. Ramrath DJ
    2. Lancaster L
    3. Sprink T
    4. Mielke T
    5. Loerke J
    6. Noller HF
    7. Spahn CM

    (2013) Visualization of two transfer rnas trapped in transit during elongation factor g-mediated translocation

    Proceedings of the National Academy of Sciences of the United States of America 110:20964–20969.

    https://doi.org/10.1073/pnas.1320387110

    • Google Scholar
92. 1. Ratje AH
    2. Loerke J
    3. Mikolajka A
    4. Brünner M
    5. Hildebrand PW
    6. Starosta AL
    7. Dönhöfer A
    8. Connell SR
    9. Fucini P
    10. Mielke T
    11. Whitford PC
    12. Onuchic JN
    13. Yu Y
    14. Sanbonmatsu KY
    15. Hartmann RK
    16. Penczek PA
    17. Wilson DN
    18. Spahn CMT

    (2010) Head swivel on the ribosome facilitates translocation by means of intra-subunit trna hybrid sites

    Nature 468:713–716.

    https://doi.org/10.1038/nature09547

    • Google Scholar
93. 1. Ren Q
    2. Au HH
    3. Wang QS
    4. Lee S
    5. Jan E

    (2014) Structural determinants of an internal ribosome entry site that direct translational reading frame selection

    Nucleic Acids Research 42:9366–9382.

    https://doi.org/10.1093/nar/gku622

    • Google Scholar
94. 1. Ren Q
    2. Wang QS
    3. Firth AE
    4. Chan MM
    5. Gouw JW
    6. Guarna MM
    7. Foster LJ
    8. Atkins JF
    9. Jan E

    (2012) Alternative reading frame selection mediated by a trna-like domain of an internal ribosome entry site

    Proceedings of the National Academy of Sciences of the United States of America 109:E630.

    https://doi.org/10.1073/pnas.1111303109

    • Google Scholar
95. 1. Rodnina MV
    2. Savelsbergh A
    3. Katunin VI
    4. Wintermeyer W

    (1997) Hydrolysis of GTP by elongation factor G drives trna movement on the ribosome

    Nature 385:37–41.

    https://doi.org/10.1038/385037a0

    • Google Scholar
96. 1. Rohou A
    2. Grigorieff N

    (2015) CTFFIND4: Fast and accurate defocus estimation from electron micrographs

    Journal of Structural Biology 192:216–221.

    https://doi.org/10.1016/j.jsb.2015.08.008

    • Google Scholar
97. 1. Rosenthal PB
    2. Henderson R

    (2003) Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy

    Journal of Molecular Biology 333:721–745.

    https://doi.org/10.1016/j.jmb.2003.07.013

    • Google Scholar
98. 1. Roy A
    2. Kucukural A
    3. Zhang Y

    (2010) I-TASSER: a unified platform for automated protein structure and function prediction

    Nature Protocols 5:725–763.

    https://doi.org/10.1038/nprot.2010.5

    • Google Scholar
99. 1. Ruehle MD
    2. Zhang H
    3. Sheridan RM
    4. Mitra S
    5. Chen Y
    6. Gonzalez RL
    7. Cooperman BS
    8. Kieft JS

    (2015) A dynamic RNA loop in an IRES affects multiple steps of elongation factor-mediated translation initiation

    eLife 4:.

    https://doi.org/10.7554/eLife.08146

    • Google Scholar
100. 1. Sahu B
     2. Khade PK
     3. Joseph S

     (2012) Functional replacement of two highly conserved tetraloops in the bacterial ribosome

     Biochemistry 51:7618–7626.

     https://doi.org/10.1021/bi300930r

     • Google Scholar
101. 1. Salsi E
     2. Farah E
     3. Dann J
     4. Ermolenko DN

     (2014) Following movement of domain IV of elongation factor G during ribosomal translocation

     Proceedings of the National Academy of Sciences of the United States of America 111:15060–15065.

     https://doi.org/10.1073/pnas.1410873111

     • Google Scholar
102. 1. Sasaki J
     2. Nakashima N

     (1999)

     Translation initiation at the CUU codon is mediated by the internal ribosome entry site of an insect picorna-like virus in vitro

     Journal of Virology 73:1219–1226.

     • Google Scholar
103. 1. Savelsbergh A
     2. Katunin VI
     3. Mohr D
     4. Peske F
     5. Rodnina MV
     6. Wintermeyer W

     (2003) An elongation factor g-induced ribosome rearrangement precedes trna-mrna translocation

     Molecular Cell 11:1517–1523.

     https://doi.org/10.1016/S1097-2765(03)00230-2

     • Google Scholar
104. 1. Schuwirth BS
     2. Borovinskaya MA
     3. Hau CW
     4. Zhang W
     5. Vila-Sanjurjo A
     6. Holton JM
     7. Cate JH

     (2005) Structures of the bacterial ribosome at 3.5 A resolution

     Science 310:827–834.

     https://doi.org/10.1126/science.1117230

     • Google Scholar
105. 1. Schüler M
     2. Connell SR
     3. Lescoute A
     4. Giesebrecht J
     5. Dabrowski M
     6. Schroeer B
     7. Mielke T
     8. Penczek PA
     9. Westhof E
     10. Spahn CM

     (2006) Structure of the ribosome-bound cricket paralysis virus IRES RNA

     Nature Structural & Molecular Biology 13:1092–1096.

     https://doi.org/10.1038/nsmb1177

     • Google Scholar
106. 1. Selmer M
     2. Dunham CM
     3. Murphy FV
     4. Weixlbaumer A
     5. Petry S
     6. Kelley AC
     7. Weir JR
     8. Ramakrishnan V

     (2006) Structure of the 70S ribosome complexed with mrna and trna

     Science 313:1935–1942.

     https://doi.org/10.1126/science.1131127

     • Google Scholar
107. 1. Sengupta J
     2. Nilsson J
     3. Gursky R
     4. Kjeldgaard M
     5. Nissen P
     6. Frank J

     (2008) Visualization of the eef2-80s ribosome transition-state complex by cryo-electron microscopy

     Journal of Molecular Biology 382:179–187.

     https://doi.org/10.1016/j.jmb.2008.07.004

     • Google Scholar
108. 1. Shaikh TR
     2. Yassin AS
     3. Lu Z
     4. Barnard D
     5. Meng X
     6. Lu TM
     7. Wagenknecht T
     8. Agrawal RK

     (2014) Initial bridges between two ribosomal subunits are formed within 9.4 milliseconds, as studied by time-resolved cryo-em

     Proceedings of the National Academy of Sciences of the United States of America 111:9822–9827.

     https://doi.org/10.1073/pnas.1406744111

     • Google Scholar
109. 1. Spahn CMT
     2. Gomez-Lorenzo MG
     3. Grassucci RA
     4. Jørgensen R
     5. Andersen GR
     6. Beckmann R
     7. Penczek PA
     8. Ballesta JPG
     9. Frank J

     (2004a) Domain movements of elongation factor eef2 and the eukaryotic 80S ribosome facilitate trna translocation

     The EMBO Journal 23:1008–1019.

     https://doi.org/10.1038/sj.emboj.7600102

     • Google Scholar
110. 1. Spahn CMT
     2. Jan E
     3. Mulder A
     4. Grassucci RA
     5. Sarnow P
     6. Frank J

     (2004b) Cryo-EM visualization of a viral internal ribosome entry site bound to human ribosomes

     Cell 118:465–475.

     https://doi.org/10.1016/j.cell.2004.08.001

     • Google Scholar
111. 1. Spiegel PC
     2. Ermolenko DN
     3. Noller HF

     (2007) Elongation factor G stabilizes the hybrid-state conformation of the 70S ribosome

     RNA 13:1473–1482.

     https://doi.org/10.1261/rna.601507

     • Google Scholar
112. 1. Spirin AS

     (1969) A model of the functioning ribosome: Locking and unlocking of the ribosome subparticles

     Cold Spring Harbor Symposia on Quantitative Biology 34:, 197–-207.

     https://doi.org/10.1101/SQB.1969.034.01.026

     • Google Scholar
113. 1. Studer SM
     2. Feinberg JS
     3. Joseph S

     (2003) Rapid kinetic analysis of ef-g-dependent mrna translocation in the ribosome

     Journal of Molecular Biology 327:369–381.

     https://doi.org/10.1016/S0022-2836(03)00146-3

     • Google Scholar
114. 1. Svidritskiy E
     2. Brilot AF
     3. Koh CS
     4. Grigorieff N
     5. Korostelev AA

     (2014) Structures of yeast 80S ribosome-trna complexes in the rotated and nonrotated conformations

     Structure 22:1210–1218.

     https://doi.org/10.1016/j.str.2014.06.003

     • Google Scholar
115. 1. Svidritskiy E
     2. Ling C
     3. Ermolenko DN
     4. Korostelev AA

     (2013) Blasticidin S inhibits translation by trapping deformed trna on the ribosome

     Proceedings of the National Academy of Sciences of the United States of America 110:12283–12288.

     https://doi.org/10.1073/pnas.1304922110

     • Google Scholar
116. 1. Taylor D
     2. Unbehaun A
     3. Li W
     4. Das S
     5. Lei J
     6. Liao HY
     7. Grassucci RA
     8. Pestova TV
     9. Frank J

     (2012) Cryo-EM structure of the mammalian eukaryotic release factor erf1-erf3-associated termination complex

     Proceedings of the National Academy of Sciences of the United States of America 109:18413–18418.

     https://doi.org/10.1073/pnas.1216730109

     • Google Scholar
117. 1. Taylor DJ
     2. Nilsson J
     3. Merrill AR
     4. Andersen GR
     5. Nissen P
     6. Frank J

     (2007) Structures of modified eef2 80S ribosome complexes reveal the role of GTP hydrolysis in translocation

     The EMBO Journal 26:2421–2431.

     https://doi.org/10.1038/sj.emboj.7601677

     • Google Scholar
118. 1. Tourigny DS
     2. Fernandez IS
     3. Kelley AC
     4. Ramakrishnan V

     (2013) Elongation factor G bound to the ribosome in an intermediate state of translocation

     Science 340:1235490.

     https://doi.org/10.1126/science.1235490

     • Google Scholar
119. 1. Valle M
     2. Zavialov A
     3. Sengupta J
     4. Rawat U
     5. Ehrenberg M
     6. Frank J

     (2003) Locking and unlocking of ribosomal motions

     Cell 114:123–134.

     https://doi.org/10.1016/S0092-8674(03)00476-8

     • Google Scholar
120. 1. van Heel M
     2. Harauz G
     3. Orlova EV
     4. Schmidt R
     5. Schatz M

     (1996) A new generation of the IMAGIC image processing system

     Journal of Structural Biology 116:17–24.

     https://doi.org/10.1006/jsbi.1996.0004

     • Google Scholar
121. 1. Voorhees RM
     2. Ramakrishnan V

     (2013) Structural basis of the translational elongation cycle

     Annual Review of Biochemistry 82:203–236.

     https://doi.org/10.1146/annurev-biochem-113009-092313

     • Google Scholar
122. 1. Voorhees RM
     2. Schmeing TM
     3. Kelley AC
     4. Ramakrishnan V

     (2010) The mechanism for activation of GTP hydrolysis on the ribosome

     Science 330:835–838.

     https://doi.org/10.1126/science.1194460

     • Google Scholar
123. 1. Wang QS
     2. Jan E

     (2014) Switch from cap- to factorless ires-dependent 0 and +1 frame translation during cellular stress and dicistrovirus infection

     PloS One 9:e103601.

     https://doi.org/10.1371/journal.pone.0103601

     • Google Scholar
124. 1. Wilson JE
     2. Pestova TV
     3. Hellen CU
     4. Sarnow P

     (2000) Initiation of protein synthesis from the A site of the ribosome

     Cell 102:511–520.

     https://doi.org/10.1016/S0092-8674(00)00055-6

     • Google Scholar
125. 1. Yamamoto H
     2. Nakashima N
     3. Ikeda Y
     4. Uchiumi T

     (2007) Binding mode of the first aminoacyl-trna in translation initiation mediated by plautia stali intestine virus internal ribosome entry site

     The Journal of Biological Chemistry 282:7770–7776.

     https://doi.org/10.1074/jbc.M610887200

     • Google Scholar
126. 1. Yang J
     2. Yan R
     3. Roy A
     4. Xu D
     5. Poisson J
     6. Zhang Y

     (2015) The I-TASSER Suite: protein structure and function prediction

     Nature Methods 12:7–8.

     https://doi.org/10.1038/nmeth.3213

     • Google Scholar
127. 1. Zhou J
     2. Lancaster L
     3. Donohue JP
     4. Noller HF

     (2013) Crystal structures of ef-g-ribosome complexes trapped in intermediate states of translocation

     Science 340:1236086.

     https://doi.org/10.1126/science.1236086

     • Google Scholar
128. 1. Zhou J
     2. Lancaster L
     3. Donohue JP
     4. Noller HF

     (2014) How the ribosome hands the a-site trna to the P site during ef-g-catalyzed translocation

     Science 345:1188–1191.

     https://doi.org/10.1126/science.1255030

     • Google Scholar
129. 1. Zhu J
     2. Korostelev A
     3. Costantino DA
     4. Donohue JP
     5. Noller HF
     6. Kieft JS

     (2011) Crystal structures of complexes containing domains from two viral internal ribosome entry site (IRES) rnas bound to the 70S ribosome

     Proceedings of the National Academy of Sciences of the United States of America 108:1839–1844.

     https://doi.org/10.1073/pnas.1018582108

     • Google Scholar

Author details

1. Priyanka D Abeyrathne

   Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States

   Contribution

   PDA, Collected and analyzed cryo-EM data, Drafting or revising the article

   Contributed equally with

   Cha San Koh and Timothy Grant

   Competing interests

   No competing interests declared.

2. Cha San Koh

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

   Contribution

   CSK, Prepared the ribosome•IRES•eEF2 complex, Built and refined structural models, Analysis and interpretation of data, Drafting or revising the article

   Contributed equally with

   Priyanka D Abeyrathne and Timothy Grant

   Competing interests

   No competing interests declared.

   "This ORCID iD identifies the author of this article:" 0000-0002-1579-0362

3. Timothy Grant

   Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States

   Contribution

   TG, Assisted with cryo-EM data processing and analyses, Drafting or revising the article

   Contributed equally with

   Priyanka D Abeyrathne and Cha San Koh

   Competing interests

   No competing interests declared.

4. Nikolaus Grigorieff

   Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States

   Contribution

   NG, Designed the project, Assisted with cryo-EM data processing and analyses, Drafting or revising the article

   For correspondence

   niko@grigorieff.org

   Competing interests

   NG: Reviewing editor, eLife

5. Andrei A Korostelev

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

   Contribution

   AAK, Designed the project, Built and refined structural models, Analysis and interpretation of data, Drafting or revising the article

   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

• 4,319

  Page views

• 987

  Downloads

• 90

  Citations

Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.