Structural Basis for Interaction of the Ribosome with the Switch Regions of GTP-Bound Elongation Factors

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1 Article Structural Basis for Interaction of the Ribosome with the Switch Regions of GTP-Bound Elongation Factors Sean R. Connell, 1,7 Chie Takemoto, 2,7 Daniel N. Wilson, 3,8 Hongfei Wang, 2 Kazutaka Murayama, 2,5 Takaho Terada, 2 Mikako Shirouzu, 2 Maximilian Rost, 1 Martin Schüler, 1 Jan Giesebrecht, 1 Marylena Dabrowski, 1 Thorsten Mielke, 4 Paola Fucini, 3 Shigeyuki Yokoyama, 2,6, * and Christian M.T. Spahn 1, * 1 Institut für Medizinische Physik und Biophysik, Charite Universitätsmedizin Berlin, Ziegelstrasse 5-9, Berlin, Germany 2 RIKEN Genomic Sciences Center, Suehiro-cho, Tsurumi, Yokohama , Japan 3 AG Ribosomen 4 UltraStrukturNetzwerk Max Planck Institute for Molecular Genetics, Ihnestrasse 73, Berlin, Germany 5 Tohoku University Biomedical Engineering Research Organization, 2-1 Seiryo-machi, Aoba-ku, Sendai , Japan 6 Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo , Japan 7 These authors contributed equally to this work. 8 Present address: Gene Center, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, Munich, Germany. *Correspondence: christian.spahn@charite.de (C.M.T.S.), yokoyama@biochem.s.u-tokyo.ac.jp (S.Y.) DOI /j.molcel SUMMARY Elongation factor G (EF-G) catalyzes trna translocation on the ribosome. Here a cryo-em reconstruction of the 70SEF-G ribosomal complex at 7.3 Å resolution and the crystal structure of EF-G-2GTP, an EF-G homolog, at 2.2 Å resolution are presented. EF-G-2GTP is structurally distinct from previous EF-G structures, and in the context of the cryo-em structure, the conformational changes are associated with ribosome binding and activation of the GTP binding pocket. The P loop and switch II approach A2660-A2662 in helix 95 of the 23S rrna, indicating an important role for these conserved bases. Furthermore, the ordering of the functionally important switch I and II regions, which interact with the bound GTP, is dependent on interactions with the ribosome in the ratcheted conformation. Therefore, a network of interaction with the ribosome establishes the active GTP conformation of EF-G and thus facilitates GTP hydrolysis and trna translocation. INTRODUCTION During protein synthesis, elongation of the nascent chain by the ribosome is steered by two G proteins, named EF-Tu and EF-G in bacteria (Wilson and Nierhaus, 2003). EF-Tu binds the ribosome as a ternary complex (EF-TuGTPaminoacyl-tRNA) and delivers the cognate aminoacyl-trna (aa-trna) to the ribosomal A site. When EF-Tu hydrolyzes the bound GTP, the aa-trna is released from the ternary complex and becomes fully accommodated into the ribosomal A site. Subsequently, the ribosome catalyzes peptide bond formation, and the newly arrived amino acid is incorporated into the nascent protein. In the presence of GTP, EF-G binds this pretranslocational state (PRE) ribosome with the trnas in the A and P sites (A- and P-tRNA) and translocates the trnas to the P and E sites (P- and E-tRNA), generating a posttranslocational state (POST) ribosome. This frees the A site and advances the ribosome by one codon on the messenger RNA (mrna). The cycle then repeats, with EF-Tu delivering a new aa-trna as dictated by the codon now exposed in the ribosomal A site. The mechanism of EF-Tu appears to follow the classical scheme of G proteins (Hilgenfeld, 1995; Yokosawa et al., 1973) in that it is active in the GTP bound form and inactive in the GDP bound form: in the GTP form, EF-Tu has high affinity for aa-trnas, which is markedly decreased upon GTP hydrolysis. Local conformational changes in the GTP binding pocket of EF-Tu, which are in turn translated into a gross conformational change in the overall structure, are responsible for the change in affinity for aatrna (Nyborg et al., 1996). Specifically, these local changes involve a restructuring of the switch I and II regions (Abel et al., 1996; Polekhina et al., 1996), which interact with the phosphate groups of the bound GTP and are named for the fact that they switch their conformations during the GTP-to-GDP transition. In contrast, the mechanism for EF-G-catalyzed translocation is not well understood and remains a controversial subject. Pre-steady-state studies (Rodnina et al., 1997; Savelsbergh et al., 2003; Wilden et al., 2006) suggested that the binding of EF-G to the ribosome induces a conformational change leading to GTPase activation, after which EF-G exists in a GDPPi state in which the GTP binding Molecular Cell 25, , March 9, 2007 ª2007 Elsevier Inc. 751

2 pocket is closed and the inorganic phosphate (Pi) is not released. A subsequent rate-limiting step, unlocking, occurs, Pi is released (suggesting that the GTP binding pocket opens), and the trna-mrna complex is moved. A modification to this pathway was presented by Pan and colleagues in which the conformational change associated with unlocking involves the movement of the P-tRNA into a P/E hybrid site while the A site trna moves afterwards (Pan et al., 2006). In an alternative model, Zavialov et al. proposed that the exchange of GDP for GTP on the ribosome promotes the mrna unlocking and translocation step, which is followed by GTP hydrolysis (Zavialov et al., 2005). Regardless of the specific model used to describe translocation, all of the models propose that both the ribosome and EF-G undergo conformational changes that are important for GTPase activation. High-resolution X-ray structures of EF-G have not been forthcoming in revealing the structural nature of these conformational changes, as they have shown that the overall conformation of EF-G is essentially similar, regardless of the nature of the bound nucleotide (Ævarsson et al., 1994; al-karadaghi et al., 1996; Czworkowski et al., 1994; Hansson et al., 2005b, 2005a; Laurberg et al., 2000). Notably, in these structures, the electron density for the switch regions, which are important for coupling G-nucleotide binding to structural rearrangements, are either generally absent or display a large degree of disorder in the X-ray structures. The discontinuity between GTP/GDP binding and structural changes in EF-G is also evident in small angle X-ray scattering experiments, showing that the overall conformation of EF-G free in solution is similar, regardless of the nature of the bound nucleotide (Czworkowski and Moore, 1997). In contrast to the free EF-G structures, mediumresolution (11 18 Å) cryo-em reconstructions revealed that, when bound to the ribosome, EF-G undergoes large-scale conformational changes (Agrawal et al., 1999; Frank and Agrawal, 2000; Spahn et al., 2004; Stark et al., 2000; Valle et al., 2003b). In these models, a relative rearrangement in the five domains of EF-G results in domain IV being inserted into the A site and either pushing the A-tRNA into the P site and/or occupying the A site after translocation in order to prevent backward slippage of the trnas (Wilson and Noller, 1998). Cryo-EM also revealed that, upon EF-G binding, the ribosome undergoes a ratchet-like subunit rearrangement (RSR) in which the subunits twist relative to one another (Spahn et al., 2001, 2004; Frank and Agrawal, 2000; Valle et al., 2003b). In particular, during the RSR, the head of the small ribosomal subunit rotates in the same direction as the trnas during translocation (Schuwirth et al., 2005; Spahn et al., 2004), potentially moving together to guide and control the translocation reaction. Since the X-ray structures suggested that nucleotide binding is not sufficient to switch the overall conformation of EF-G, a concerted effort between EF-G and the ribosome is likely to be involved in promoting these structural changes. Here we present a cryo-em structure of the 70SEF- GGMPPNP complex from Thermus thermophilus at 7.3 Å resolution, as well as a 2.2 Å structure of T. thermophilus EF-G-2GTP, a homolog of EF-G. The subnanometer resolution of the cryo-em reconstruction allows the observation of the switch I region of EF-G and the characterization of its contacts within the ribosomeef-g complex. The EF-G-2GTP structure reveals conformational changes in EF-G-2 resulting from GTP binding. The overall conformation of EF-G-2GTP, with the presence of an ordered switch I region, bears similarities to the cryo-em reconstruction of EF-GGMPPNP, indicating that EF-G- 2GTP has been crystallized in a conformation related to the active structure rather than the free solution structure represented by the previous EF-G structures. Collectively, the crystal structure and cryo-em reconstruction provide a model to explain how coordinated communication between the ribosome and EF-G regulates the GTPase and translational activities of EF-G. RESULTS Cryo-EM Reconstruction of the 70SEF-GGMPPNP Complex from T. thermophilus For image processing, we recorded micrographs of a T. thermophilus 70SEF-G complex in which EF-G was stalled on the ribosome, via the nonhydrolyzable GTP analog GMPPNP. Although the density for EF-G could be directly observed in the initial cryo-em maps, the substoichiometric occupancy (see the Experimental Procedures) resulted in a fragmented appearance of EF-G, especially as the reconstruction progressed toward higher resolution. In order to solve this problem and to obtain a more homogeneous subset of particles, we employed a multireference 3D projection refinement procedure (Penczek et al., 2006) and selected a subset of particles bound by EF-G. Thus we selected 77,038 out of a total of 362,361 projections and obtained a final model (Figure 1) with a resolution of 7.3 Å, according to the FSC curve using the 0.5 cutoff criterion (see Figure S1 in the Supplemental Data available with this article online). In accordance with the resolution estimate, the grooves of the RNA helices are easily distinguished, the a-helical secondary structure within EF-G and the ribosomal proteins is observed, and in some cases the unstructured peptide chains corresponding to the extended tails of ribosomal proteins are resolved (Figure S2). In agreement with previous studies of the E. coli ribosome (Spahn et al., 2001; Frank and Agrawal, 2000; Valle et al., 2003b), the T. thermophilus 70SEF-GGMPPNP complex undergoes RSR (Supplemental Results). Similar to X-ray studies of T. thermophilus ribosomes (Yusupov et al., 2001), our reconstruction also contains a trna that copurified with the 70S ribosomes (Figure 1; green density); however, here it is located in the P/E site rather than the E site (Supplemental Results). Therefore, biochemically, our complex is similar to those of previous studies, in which EF-G was trapped by a nonhydrolyzsable GTP analog on ribosomal 752 Molecular Cell 25, , March 9, 2007 ª2007 Elsevier Inc.

3 Figure 1. Cryo-EM Reconstruction of a T. thermophilus 70SEF-GGMPPNP Complex The cryo-em reconstruction is shown from a side (left) and top (right) view. The 30S subunit is colored yellow, the 50S subunit is blue, the P/E trna is green, and EF-G is red. Major landmarks are indicated in the figure where the L7/L12 stalk base, central protuberance, head, beak, and shoulder are abbreviated as SB, CP, h, b, and sh, respectively. Fragmented density for ribosomal proteins L9 and L7/L12 and the N- and C-terminal domains (NTD and CTD) of L11 are also indicated. complexes lacking an A-tRNA and containing a deacylated P-tRNA (Valle et al., 2003b). A few studies attempted to visualize a complex containing EF-G together with an aminoacylated A-tRNA (Agrawal et al., 1999), but it was later shown that the samples were heterogeneous, and the EF-G and trnas were present in different ribosome populations (Penczek et al., 2006). Characterization of EF-G-2 In half of the bacterial genomes sequenced to date, a gene for EF-G-2, an EF-G-like protein, has been identified. In the genome of T. thermophilus HB8, a 658 amino acid open reading frame named EF-G-2, with 34% identity and 56% similarity to EF-G (Figure S3), was identified. Overexpressed and purified EF-G-2 shows a detectable intrinsic GTPase activity in the absence of ribosomes (Figure 2A). In contrast, the ribosome-dependent GTPase assays demonstrate that EF-G-2 and EF-G are similarly activated by T. thermophilus ribosomes (Figure 2B). In in vitro poly(phe) synthesis, EF-G-2 displays slightly lower activity than EF-G but still exhibits a significant activity when compared to control reactions (Figure 2C). These results reveal that EF-G-2 is a valid model for studying EF-G function, despite the sequence differences. X-Ray Structure of EF-G-2GTP EF-G-2 in complex with GTP was crystallized, and its structure was solved at 2.2 Å resolution by the multiwavelength anomalous dispersion method (Table 1). In the EF- G-2 structure, the switch I region (Arg38-Arg63 in EF-G-2), which is largely disordered in all of the reported EF-G structures, has been modeled to fold into a single turn of a3 10 helix (helix A 1 0 ) and an a helix (helix A 1 00 ; red cartoon ribbon in Figure 2D). EF-G-2 has overall dimensions of Å and consists of five domains with similar folds to those of EF-G (Laurberg et al., 2000; Figure 2D) such that each domain superimposes well on its EF-G counterpart with an RMSD of Å for the Ca atoms of homologous residues. The relative arrangement of domains I and II in EF-G-2 is similar to that seen in the previous EF-G structures (Ævarsson et al., 1994; al-karadaghi et al., 1996; Czworkowski et al., 1994; Hansson et al., 2005a, 2005b; Laurberg et al., 2000), whereas domain III and the domain IV/V block are rearranged independently, resulting in an overall displacement of the tip of domain IV by approximately 20 Å. The independent movement of domain III (Movie S1) is, in part, facilitated by conformational changes in the linker regions joining domain III to domains II and IV. Furthermore, the domain III reorientation is accompanied by a change in the interface between domains III and V, which could contribute to the displacement of the domain IV/V block associated with translocation (Movie S1). The observed differences in the domain arrangement of EF-G-2 as compared to previous EF-G structures reflect the GDP-to-GTP transition and are reminiscent of the situation in EF-Tu, where the state of G-nucleotide binding dramatically affects the conformation of the protein (Nyborg et al., 1996). The Ribosome-Bound Structure of EF-G A model for T. thermophilus ribosome-bound EF-G (rbef- G) was generated by docking individual domains from either the structure representing the H573A EF-G mutant bound to GDP (Laurberg et al., 2000) or that of EF-G-2 bound to GTP into our cryo-em map (Supplemental Results). For the most part, the individual domains from both crystal structures fit easily into the density, indicating the absence of major intradomain conformation changes Molecular Cell 25, , March 9, 2007 ª2007 Elsevier Inc. 753

4 Figure 2. GTPase Activity and Crystal Structure of EF-G-2 (A and B) Assays of the intrinsic GTPase (without ribosomes, [A]) and the ribosome-dependent GTPase (B) of EF-G (blue triangles) and EF-G-2 (red circles) from T. thermophilus were performed at 65 C by monitoring the release of the g-phosphate (Pi). The reaction mixture (20 ml) contained 0.5 mm [g- 32 P]-GTP. In (A), 0.5 mm EF-G or EF-G-2 was used without ribosomes, and the Pi released in the absence of elongation factor was subtracted as background. In (B), 0.05 mm EF-G or EF-G was used and the Pi released in the presence of ribosomes, but the absence of elongation factor was subtracted as background. The reaction time was 8 min. (C) Poly(U)-dependent poly(phe) synthesis assays performed at 60 C. Each reaction mixture (10 ml) contains 3.75 pmol [ 14 C]-Phe-tRNA, 0.25 pmol ribosome, and 2.5 pmol of EF-G (blue triangles) and EF-G-2 (red circles) or no elongation factor (black squares). Error bars (A C) represent the mean ± standard deviations of repeated measurements: {n 1 S(x Sx 2 /n) 2 } 1/2. Detailed conditions are described in the Supplemental Experimental Procedures. (D) Stereo representation of the GTP-bound EF-G-2 is shown as a ribbon colored by domain: domains I, II, III, IV, and V are colored green (with the G 0 domain highlighted in olive green), blue, cyan, burgundy, and orange, respectively. Switch I is colored red, and helices B 1 and B 1 0 in switch II are yellow. The characteristic helix E 1 0 between domains I and II is colored magenta. Mg 2+ and GTP are shown as a white sphere and as orange sticks, respectively. (Figure 3A). The alignment of secondary structure elements, such as a helices, in the crystal structure with cylindrical density in our electron density maps augmented the accuracy of this docking (Figure 3A; also see Figures 4A 4F for examples). The model of T. thermophilus rbef-g is similar to that determined previously; however, there are some differences (Supplemental Results). The most significant differences are seen in the placement of domain V, which in previous cryo-em reconstructions (Valle et al., 2003b) used a different surface to interact with the ribosomal stalk base (H43/44) than that seen in the current T. thermophilus rbef-g model (Figure S8). 754 Molecular Cell 25, , March 9, 2007 ª2007 Elsevier Inc.

5 Table 1. Structure Determination Statistics X-Ray Data Native Peak Edge High Remote Space group C2 C2 C2 C2 Unit cell (Å) a b c b ( ) Resolution (Å) Wavelength (Å) Number of observations 151, , , ,627 Number of unique reflections a 39,997 (3964) 24,103 24,081 24,071 Completeness (%) (100.0) (99.9) (99.9) (99.9) <I>/s (I) 14.5 (3.8) 12.8 (4.1) 13.9 (4.1) 14.8 (4.5) R sym (%) b 9.3 (38.3) 11.6 (35.3) 10.8 (35.1) 10.1 (31.3) Refinement R c cryst (%) 19.7 R d free (%) 24.1 Rmsd from Standard Stereochemistry Bond lengths (Å) Bond angles ( ) 1.2 Ramachandran Plot Statistics Most favored regions (%) 91.8 Additional allowed regions (%) 8.0 Generously allowed regions (%) 0.2 Disallowed regions (%) 0.0 a Numbers in parentheses represent values in the highest resolution shell (native Å, SeMet Å). b R sym = SjI j <I>j/SI j, where I j is the observed integrated intensity, <I> is the average integrated intensity obtained from multiple measurements, and the summation is over all observed reflections. c R cryst = SjjF obs j jf calc jj/sjf obs j.f obs and F calc are observed and calculated structure factor amplitudes, respectively. d R free calculated with randomly selected reflections (5%). Interestingly, in the eukaryotic eef280s structure (Spahn et al., 2004), the modeled interaction between domain V and the ribosome is the same as that observed in T. thermophilus, suggesting the universality of the interaction. When the crystal structures of EF-G are compared to the rbef-g model, it is obvious that rbef-g is more similar to EF-G-2GTP, in terms of the overall domain arrangement, than to the previous EF-GGDP crystal structures (Figures 3B and 3C). In particular, the EF-G-2 structure depicts the displacement of domain IV that is characteristic of rbef-g, although it is still not in a fully extended conformation (Figure 3C). Furthermore, the cryo-em study shows that when EF-G is bound to the ribosome, domain III of EF-G rotates relative to domains I and II, altering the interactions between domain III and the switch regions in domain I (Supplemental Results and described below). Virtually the same relative orientation of domains I and III is observed in the EF-G-2 crystal structure (Figure 3C). This indicates that the conformational changes seen in the EF-G-2GTP structure relative to the EF-GGDP structures are biologically relevant and represent an important step in the pathway to adopt the rbef-g conformation. The ability of EF-G-2 to adopt this conformation in the absence of the ribosome may be a particular characteristic of EF-G-2 that distinguishes it from EF-G, because previous structures of EF-G in the GTP form are structurally similar to the GDP form. Alternatively, interactions within the EF-G-2 crystal may substitute the role of the ribosome, and therefore the previous EF-GGMPPNP structure may not have the correct crystal form to stabilize the ribosome-bound conformation. Molecular Cell 25, , March 9, 2007 ª2007 Elsevier Inc. 755

6 Figure 3. The Ribosome-Bound Structure of EF-G (A) The result of docking, as rigid bodies, the five domains of EF-GGDP (Laurberg et al., 2000) into the cryo-em reconstruction (gray mesh). The rbef-g structure depicted in (A) is superimposed on domain I of the (B) EF-GGDP (1FNM; gray) and (C) EF-G-2GTP (gray) X-ray structures to highlight the similar domain arrangement of rbefg and EF-G Molecular Cell 25, , March 9, 2007 ª2007 Elsevier Inc.

7 Figure 4. Fitting of the EF-G Crystal Structure into the Cryo-EM Reconstruction (A) The interaction of rbef-g with SRL of the large ribosomal subunit. (B) In the cryo-em map (mesh), additional density is observed in the vicinity of the GTP binding pocket. This density can accommodate the distal end of switch 1 (violet) when it is positioned such that residues Thr60, Thr61, and Asp50 (of EF-G-2; sticks) are placed relative to the GTP molecule as observed in the EF-G-2 crystal structure. (C) Interactions of domain II with h5 of the 30S subunit. (D) The interaction of domain III with S12. (E) The interaction of domain IV with h44 of the 30S subunit and H69 of the 50S subunit. H69 appears to undergo a conformational change to accommodate the RSR (Supplemental Results), which appears to distort the loop of H69 such that, in this model, nucleotide 1913 (light pink) is not accounted for in the electron density map. (F) Interaction of domain V with the large ribosomal subunit. Molecular Cell 25, , March 9, 2007 ª2007 Elsevier Inc. 757

8 Molecular Interactions between EF-G and the Ribosome The overall interaction pattern between EF-G and the 70S ribosome from T. thermophilus in the present cryo-em structure is similar to that seen with E. coli EF-G and yeast eef2 (Agrawal et al., 1999; Frank and Agrawal, 2000; Spahn et al., 2004; Valle et al., 2003b). However, due to the subnanometer resolution of the present cryo-em structure, the contacts can be localized with a higher degree of certainty (summarized in Table S1 and Figure 4). Domain I is bound below the L7/L12 stalk, where it interacts with the sarcin-ricin loop (SRL; H95) on the 50S ribosomal subunit and helix 14 (h14) of the 30S ribosomal subunit (Figures 4A and 4B). Domain II contacts the 30S subunit in the region of helix 5 (h5) and h15 (Figure 4C), while domain III interacts with the 30S ribosomal subunit protein S12 (Figure 4D). This interaction with S12 is likely to be functionally significant, as Gavrilova et al. (1974) and Cukras et al. (2003) demonstrated a role for S12 in promoting translocation. Domain IV of EF-G penetrates into the A site such that it would overlap with the stem of the anticodon arm of the A-tRNA and interact with helices 44 (h44) and 69 (H69) of the 30S and 50S subunits, respectively (Figures 4E). On the 50S subunit, the base of the ribosomal L7/L12 stalk mediates contacts with domain V of EF-G (Figures 4F). The cryo-em map provides a detailed view of the GTP binding domain (domain I) of EF-G and its interactions with the ribosome. In general, the GTP binding pocket of EF-G comprises five typical motifs (G1 G5) for guaninenucleotide recognition (Bourne et al., 1991). The P loop (including the G1 motif) and the switch I and II regions (including the G2 and G3 motifs, respectively) monitor the phosphate groups, while the G4 and G5 motifs are involved in recognition of the guanosine. Interestingly, these conserved elements are close to the sites of contact between the ribosome and EF-G. As seen in Figure 4A, there is a strong fusion of density between the P loop, the switch II region, and the SRL, which has been suggested to be a candidate for stimulating the ribosome-dependent GTPase of EF-G (Chan et al., 2004). The tight packing of the SRL between domains I, III, and V (Figure 4A and Table S1) likely requires local conformational changes in both the SRL and EF-G to resolve the close contacts seen in our model. Switch I can also be localized in this study, because it is ordered in the EF-G-2GTP structure, and when it is aligned to the cryo-em reconstruction, the switch I region coincides with the additional density seen in our map (Figure 4B). The fitting seen in Figure 4B would maintain interactions between the distal region of switch I, the guanine nucleotide, and the a helix B 3, as seen in the EF-G-2 crystal structure (see below). In fact, the cryo-em reconstruction shows a strong fusion of density between switch I (helix A 1 00 ) and helix B 3 in domain III (Figure 4B), reinforcing the similarity in the conformation of domains I, II, and III observed among the EF-G-2GTP and rbef-g structures. It is clear, however, that residues 34 46, including the helix A 1 0, are not well within the cryo-em map. These residues likely undergo a conformational change such that they would fit into the density that fuses near nucleotide 344 of h14 in the 30S ribosomal subunit (Figure S4). In previous cryo-em reconstructions of EF-G and EF-Tu, switch I was suggested to interact with the SRL (Agrawal et al., 1999; Valle et al., 2003a), and although such an interaction cannot be excluded, it is not observed in the present reconstruction. This difference may reflect the fact that, in many of the previous studies, the factor was in the GDP state, whereas we describe a complex in the GTP state. In the cryo-em reconstruction presented here, the ribosome has undergone RSR. In this regard, the interactions formed by domains I and III of rbef-g and the 30S subunit (S12 and h14) may be transient and dependent on the relative ribosomal subunit arrangement. For example, helix 14, which contacts switch I, would move by approximately 8Åin the absence of the RSR. This is consistent with the fact that domain III and the switch I region of domain I are relatively flexible elements, as indicated by their partial disorder in several previous X-ray structures of EF-G (Ævarsson et al., 1994; al-karadaghi et al., 1996; Czworkowski et al., 1994; Hansson et al., 2005a, 2005b; Laurberg et al., 2000). This also implies that rbef-g can form a transient or flexible interaction surface with the 30S subunit, which is apparently necessary for the coordinated movement during the RSR (Frank and Agrawal, 2000; Spahn et al., 2001, 2004). Conformations of the Switch I and II Regions in EF-G-2GTP In the EF-G-2GTP structure, the switch I region folds into a single turn of a 3 10 helix (helix A 1 0 ) and an a helix (helix A 1 00 ), as shown in Figure 2D. Part of the switch I region (residues Tyr51 Thr65 in EF-G-2, including the helix A 1 00 ) is remarkably similar to the homologous region of the structure of the EF-Tu ternary complex (Nissen et al., 1995) (Figure 5A). In contrast, residues Arg38 Asp50, encompassing the helix A 1 0, are in a different location (Figure 5E). Although these differences may be due to crystal contacts, they are likely to be functionally significant, as (1) previous experiments have shown that the switch I regions from EF-G and EF-Tu are not interchangeable (Kolesnikov and Gudkov, 2002) and (2) the present cryo-em reconstruction suggests that this region in EF-G is responsible for communicating signals from the 30S subunit in the RSR state (Figure 4B). As shown in Figure 5B, a hydrophobic core is formed among the helix A 1 00 (switch I), the amino terminus of a helix B 1 (switch II), and a helix B 3 of domain III. This core in the EF-G-2GTP structure is formed through CH-p interactions between His58 (switch I), Leu431 (helix B 3 ), and Tyr84 (switch II) and between Val62 (switch I) and Phe87 (switch II). In addition to the hydrophobic interactions, there is a putative hydrogen bond between His58 (switch I) and Glu438 (the helix B 3 ) as well as a hydrogen-bonding network among Glu54 (switch I), Asp442 (helix B 3 ), and Arg439 (helix B 3 ), as shown in Figure 5C. This interaction 758 Molecular Cell 25, , March 9, 2007 ª2007 Elsevier Inc.

9 Figure 5. Conformational Changes in Switch I and II Seen by X-Ray Crystallography (A) A backbone alignment of EF-G-2GTP (blue) and EF-TuGMPPNPtRNA (1TTT) complex with domain I. EF-Tu and trna are colored in gray and orange. Switch I of EF-Tu and the Mg 2+ are shown in magenta ribbon and green spheres, respectively. Coloring of EF-G-2 is the same as in Figure 2D. (B and C) The hydrophobic core and hydrogen-bonding interactions between helix B 3 of domain III (cyan) and the switch I (red) and II (yellow) regions are shown with the interacting residues indicated in the panel. The distances between H58-L431, L431-84, and V62-F87 are approximately Å. Dotted lines are putative hydrogen bonds (<3 Å). The magnesium ion is represented by a blue sphere. (D) The coordination of the GTP molecule and the Mg 2+ ion (green sphere) is shown with the interacting residues in the P loop (G1 motif; yellow), the switch I (G2 motif; red), and switch II (G3 motif; magenta) regions and G4/5 motifs (pink) indicated in the panel. The electron density (jf o j jf c j map contoured at 3 s) corresponding to GTP molecule is shown. Water molecules coordinating the Mg 2+ and the g-phosphate are colored red. Dotted lines are putative polar contacts. (E) The P loop (green) and the switch I (red) and switch II (yellow) regions of EF-G-2GTP that are important for GTP binding/hydrolysis are shown superimposed on the corresponding regions from EF-TuGMPPNP (gray ribbon, 1TTT) and EF-GGDP (1FNM, cyan). Important residues are drawn as sticks and identified in the figure. The active site waters as seen in the EF-Tu and EF-G-2 structures are drawn as gray and blue spheres, respectively. The Mg 2+ and the interacting waters are colored green and red, respectively. network suggests that the ordering of switch I and the rearrangement of switch II in the EF-G-2GTP structure establishes a surface to promote the interaction with the helix B 3 of domain III. In EF-Tu, GTP binding promotes a rearrangement in the switch I and II regions and forms an interaction surface for the aa-trna (Nyborg et al., 1996). As discussed above, this interaction with domain III is also observed in the cryo-em structure of rbef-g and is therefore functionally relevant. Activation of the GTP Binding Pocket The EF-G-2GTP electron density map unambiguously shows the GTP nucleotide and the magnesium ion coordinated by the five typical motifs (G1 G5) for Molecular Cell 25, , March 9, 2007 ª2007 Elsevier Inc. 759

10 guanine-nucleotide recognition (Figure 5D) (Bourne et al., 1991). The backbone conformation of the P loop in EF-G- 2GTP is similar to that seen in the EF-Tu and EF-G crystal structures. A significant difference observed in the P loop is observed in the side chain of Lys22 (Lys25 in EF-G), which in EF-G-2 is positioned to interact with both the b- and g-phosphates, similar to that seen in all of the structurally characterized ribosomal GTPases except EF-G (discussed in Hansson et al. [2005a]). In the EF-G and eef2 structures (Ævarsson et al., 1994; Czworkowski et al., 1994; al-karadaghi et al., 1996; Laurberg et al., 2000; Hansson et al., 2005a., 2005b; Jorgensen et al., 2003), the equivalent lysine residue is pointed toward the switch II region (Figure 5E). This is notable, as this lysine residue has been suggested to be important for GTP/ GDP binding and GTP hydrolysis (Hansson et al., 2005a; Hwang et al., 1989). Therefore, the reordering of the P loop and the switch II region seen in the EF-G-2GTP structure represents a step in the activation of the GTP binding pocket. In the switch II region of EF-G-2GTP, the amino terminus of the helix B 1 is extended by three residues (Gly85-Asp86-Phe87), and the axis of the helix B 1 is shifted by approximately 20, as compared to the EF-G crystal structures (Figure 5E, compare cyan and yellow ribbons). Therefore, the conformation of the switch II region in EF-G-2 is different from that in the previous EF-G structures (Czworkowski et al., 1994; Laurberg et al., 2000; Hansson et al., 2005a; Hansson et al., 2005b) but similar to that in the EF-TuGMPPNP structure (Nissen et al., 1995). The subsequent steps in the activation of the GTP binding pocket of EF-G-2 result from the ordering of the switch I region. This region participates in the coordination of the active site magnesium ion and interacts with the oxygen atoms of the g-phosphate (Figures 5D and 5E). The ordering of switch I helps coordinate the magnesium ion by positioning Thr61 and Asp50 to make direct and indirect (via a water bridge) interactions with the ion, respectively (Figure 5E). Furthermore, Thr60 is positioned to interact directly with an oxygen atom of the g-phosphate (Figure 5D). Residues equivalent to Thr60 and Thr61 in EF-Tu have been shown to be important for the GTPase activity in EF-Tu (Krab and Parmeggiani, 1999) by forming interactions that may stabilize transition states during GTP hydrolysis. Therefore, the ordering of the switch I region positions key residues required for stabilization of reaction intermediates and represents a second step toward activation of the GTP binding pocket of EF-G-2. In both EF-TuGMPPNP (Nissen et al., 1995) and EF- G-2GTP, an active site water molecule is positioned by a potential hydrogen bond network with similar residues in the switch I and II regions for an inline attack of the g-phosphate (Figure 5D). In the well-studied GTPase, Ras, it has been proposed that a water molecule is activated when the g-phosphate (of the GTP substrate), acting as a general base, accepts the proton from the water molecule (Langen et al., 1992). Gln61 in the Ras protein then stabilizes the accumulating negative charge by providing a hydrogen bond to the nucleophilic water (Langen et al., 1992). Similarly, in EF-Tu, His84 (equivalent to Gln61 of Ras) is believed to interact with the active site water. However, one difference with respect to Ras lies in the fact that, in the EF-Tu structures, His84 has not generally been seen to interact with the water, which led to the proposal that a conformational change, similar to that seen in the EF-TuGDPaurodox structure (Vogeley et al., 2001), is needed for His84 to interact with the water molecule. As seen in Figure 5D, the homologous residue (Tyr84) in EF-G-2 is near the water molecule but is not correctly positioned to interact with it. This suggests that, despite the conformational changes in the switch I and II regions, which lead to a general activation of the GTP binding pocket, EF-G-2 is not completely prepared for GTP hydrolysis, and therefore we consider it to be a precatalytic state. This final activation step probably requires input from the ribosome. The SRL seems to be the only ribosomal element close enough to directly contribute a reactive group to the GTPase reaction. However, the SRL or other ribosomal elements (L11, L7/12, H42/43/44) that interact with EF-G may indirectly stabilize the conformation of the GTP binding pocket that promotes the GTPase reaction. This could include, for example, displacing the above-mentioned Tyr84 residue in EF-G-2 so that it can stabilize the nucleophilic water during the hydrolysis reaction. DISCUSSION We have presented here the X-ray structure of T. thermophilus EF-G-2GTP and a subnanometer resolution cryo- EM structure of T. thermophilus EF-GGMPPNP bound to the 70S ribosome. A comparison of both structures reveals a striking similarity in their overall domain arrangement (Figure 3C). This demonstrates that the X-ray structure of EF-G-2GTP provides atomic-resolution information about the active GTP-bound state, unlike the previous X-ray structures of EF-G, which most likely represent the free solution structure (Ævarsson et al., 1994; Czworkowski et al., 1994; al-karadaghi et al., 1996; Laurberg et al., 2000; Hansson et al., 2005a, 2005b). Analyses of the GTP binding pocket in the EF-G-2GTP structure revealed that many of the conformational changes distinguishing it from the previous EF-GGDP structures are similar to the changes associated with the transition between the GTP- and GDP-bound states of EF-Tu. Furthermore, the formation of a transient binding surface by conformational changes in the switch I and II regions is a conserved characteristic; in EF-Tu, this surface interacts with the trna, whereas in EF-G-2, it forms an interface with domain III. Crosstalk with the Ribosome Is Involved in GTP Binding/GTPase Activation of EF-G The involvement of the switch I and II regions in forming an interface with domain III is supported by both the cryo-em reconstruction and the crystal structure (Figures 2D and 760 Molecular Cell 25, , March 9, 2007 ª2007 Elsevier Inc.

11 Figure 6. Ribosomal Elements Interacting with EF-G Domains I, III, and V The ribosomal elements (labeled in figure) interacting with domains I, III, and V of EF-G are shown in (A), whereas in (B) the network of interactions that stabilize the switch regions and domain III is indicated. (C) A model summarizing the steps leading to GTPase activation on EF-G and subsequent stable translocation of the trnas. The reaction scheme begins with the PRE ribosome (state A), which is proposed to exist in a dynamic equilibrium in which some ribosomal particles display the RSR (red outline) and others exist in the ground state (unratcheted conformation, black outline). In the ratcheted state, the trnas would be in hybrid states. Isolated EF-G would bind the PRE ribosome and stabilize the ribosomes in the ratcheted state (state B). When EF-G binds this state, an interaction network with the 30S subunit would stabilize the GTP conformation of the switch regions (state B, represented by the dark green and elongated corner of domain I). Furthermore, the surface formed by conformational changes in the switch regions as well as interactions with S12 would promote the rotation of domain III (state B). This, in turn, would shift domains IV and V into an extended conformation (state C). It should also be noted that the order of the changes illustrated schematically in state B and state C is unknown and that they are hypothetical states. State C would be highly labile, and the release of the Pi moiety would allow the switch I and II regions to snap back to their relaxed conformation (state D). This would release the hold domain I had on domain III, allowing it to also relax, and would generally destabilize the ratcheted conformation of the ribosome (state D). Conformational changes in the ribosome that accompany EF-G action for example, changes in the L7/L12 stalk regions (Seo et al., 2006; Datta et al., 2005) have not been included in the model for simplicity. 4B). The coming together of these elements in the EF-G-2 structure is dependent on the ordering of switch I, the conformational changes in switch II, and the rotation of domain III relative to that seen in the EF-GGDP structure (Laurberg et al., 2000). These conformational changes result from the fact that GTP is bound and that interactions with the g-phosphate stabilize the switch regions. Additionally, in the crystal, contacts with neighboring molecules may contribute to the stabilization of switch I and the rotated conformation of domain III. The cryo-em reconstruction suggests that, in a biological context, the ribosome plays this role: switch I is stabilized by interactions with h14 on the 30S subunit (Figure 4B), the rotation of domain III is stabilized by contacts with the 30S ribosomal protein S12 (Figure 4D), and the conformational change in switch II could be influenced by interactions with the SRL (Figure 4A). In this regard, it appears that a network of ribosomal interactions (Figure 6B) is established to alter the conformation of EF-G and ultimately influence the state of the GTP binding pocket. Furthermore, in the cryo-em reconstruction, the ribosomal subunits are in a ratcheted conformation (Supplemental Results), and therefore this network of interactions can also be regulated by the relative arrangement of the ribosomal Molecular Cell 25, , March 9, 2007 ª2007 Elsevier Inc. 761

12 subunits. A role for the RSR in regulating EF-G GTPase is indicated by the fact that only an unlocked ribosome (PRE state) one that can undergo the RSR can stimulate GTP hydrolysis by EF-G (Zavialov and Ehrenberg, 2003). Therefore, this provides a means to ensure that EF-G productively interacts with the ribosome at the correct step in the elongation cycle and explains the synergism between EF-G and EF-Tu (Mesters et al., 1994). A Structural Model for Ribosomal Translocation The combined X-ray crystallography and cryo-em models presented here are congruent with the various models to explain translocation based on biochemical experiments (Savelsbergh et al., 2003; Seo et al., 2006; Pan et al., 2006; Zavialov et al., 2005) and provide a structural basis for many of the phenomena observed in these biochemical experiments, particularly the activation of GTPase activity on EF-G. Despite the fact that our ribosomal complex lacks authentic translocation substrates (A-tRNA), it is relevant for analyzing the ribosome-dependent GTPase activity, as vacant ribosomes are as efficient as authentic PRE complexes at stimulating EF-G-dependent GTPase (Rodnina et al., 1997). Moreover, in terms of their GTPase activity, EF-G and EF-Tu act synergistically on vacant ribosomes (Mesters et al., 1994). It has been proposed that, on the ribosome, EF-G has a higher affinity for GMPPNP than GDP, and this is important for promoting guanine nucleotide exchange (Zavialov et al., 2005). Although Wilden and colleagues believe that guanine nucleotide exchange on EF-G predominately occurs before ribosome binding, they also showed that nucleotide binding by EF-G is stabilized on the ribosome (mant-gdp by 30-fold and mant-gmppnp by 10,000- fold) and proposed that this reflects a closing of the GTP binding pocket (Wilden et al., 2006). Our structures suggest that the closing of the GTP binding pocket corresponds to the conformational changes in the switch region (Figure 6C, state B) that fold around and establish key interactions with the phosphate groups, thus explaining the increased stabilization of the tri- rather than the diphosphate nucleotide. In this conformation, the switch regions exist in a high-energy state, which has been described as a loaded spring, stabilized by interaction with the phosphate groups (Vetter and Wittinghofer, 2001). Subsequently, GTP would be hydrolyzed, possibly stimulated by ribosomal interactions, but Pi would be retained. In this context, fast GTPase, before the actual translocation reaction (Rodnina et al., 1997; Seo et al., 2006), might commit EF-G to a productive forward reaction. When Pi is released, the switch regions would relax due to the loss of contacts with the g-phosphate, allowing domain III to return to the unrotated state because the interaction surface would be disrupted (Figure 6C, state D). This, in turn, could destabilize the ratchet conformation of the ribosome and allow the 30S subunit to reset (Figure 6C, state D). An interesting proposal in the model seen in Figure 6 (state A) is that the ability to undergo RSR may be an inherent quality of the PRE ribosome and that EF-G binding would only serve to stabilize the ratcheted state of the ribosomal subunits through the network of interactions formed with the 30S subunit (Figures 6B and 6C, state B). In this regard, the ratcheted state of the subunits in eukaryotic 80S ribosomes is known to be stable in the absence of any elongation factor (Spahn et al., 2004), and the observation of hybrid sites, by chemical probing in the absence of EF-G, suggests that this ability could be conserved in bacteria (Moazed and Noller, 1989). Although the ratcheted state has not been observed by cryo-em for PRE 70S ribosomes (Valle et al., 2003b), a dynamic exchange of classical and hybrid trna binding has been observed in single-molecule FRET experiments (Blanchard et al., 2004). Additionally, in the proposed model of translocation, the ribosome undergoes RSR, but EF-G also functions essentially as a molecular ratchet. In the GTP form of EF-G, ribosome-induced conformational changes promote the rotation of domain III of EF-G, which in turn pushes domain IV into the A site and actively or passively facilitates trna movement (Figure 6C, state C). After phosphate release, domain III relaxes, but during the movement of domain III in the reverse direction, there is no coupling to the domain IV movement (Figure 6C, state D). This results in A site occupation by domain IV of EF-G, when the ribosome changes from the transition state exhibiting the RSR to the ground state and prevents reverse translocation of the trnas (Figure 6C, state D). The resulting POST complex is locked by the peptidyl-trna in the P site, preventing the stable association of EF-G and leading to the dissociation of the factor. EXPERIMENTAL PROCEDURES Cryo-EM of EF-G-70S Complexes Recombinant EF-G was purified (Blank et al., 1995) and was bound to 70S ribosomes in the presence of GMPPNP in a reaction containing 1 mm 70S ribosomes, 10 mm EF-G, 300 mm GMPPNP, 10 mm HEPES-KOH (ph 7.8), 10 mm Mg acetate, 60 mm NH 4 Cl, and 6 mm b-mercaptoethanol. The occupancy of EF-G in the complexes was about 60% 70%, as judged by a centrifugal binding assay (Sharma et al., 2005). The complex was subsequently frozen on quantifoil grids by using a Vitrobot (FEI) device and imaged under low-dose conditions. The resulting data were refined using standard procedures with the SPIDER software package (Frank et al., 1996). This included a multireference 3D projection refinement procedure (Penczek et al., 2006) in which, after generating and refining a single volume, subsequent models (low-pass filtered E. coli 70S ribosome) were introduced as seeds to increase the number of references until stable subpopulations were obtained (Supplemental Experimental Procedures). In the last refinement steps, matched filters were used to enhance high frequencies in the reference model. The final reconstructions had a resolution of 7.3 Å using the 0.5 FSC criteria. Automated docking of the ribosomal and EF-G X-ray structures (2j00, 2j01, 1YL3, 1YL4, 1WDT, 1FNM) was performed with the Situs software package (Wriggers et al., 1999). EF-G-2 Structure Determination Recombinant EF-G-2 was expressed as both native and SeMet derivatives (Supplemental Experimental Procedures), which were 762 Molecular Cell 25, , March 9, 2007 ª2007 Elsevier Inc.

13 crystallized by the hanging-drop vapor diffusion technique at 20 C. The reservoir solution consisted of 30 mm magnesium acetate, 18% isopropanol, and 30 mm MES (ph 6.0). Both native and MAD datasets were collected. The MAD data set was collected at three wavelengths up to 2.6 Å resolution. The structures were refined to an R factor of 19.7% (R free = 24.1) at 2.2 Å resolution using the native data. Detailed procedures of the structure determination are described in the Supplemental Experimental Procedures. GTPase Activity Assay Activities of elongation factors were determined by following the amount of phosphate released (Dasmahapatra and Chakraburtty, 1981). The reaction mixture (20 ml) consisted of 20 mm HEPES-/KOH (ph 7.6), 10 mm MgCl 2, 50 mm NH 4 Cl, 5 mm spermine, 4 mm 2-mercaptoethanol, and 0.5 mm [g- 32 P]-GTP (PB10244; diluted to cpm/pmol). The reactions were performed at 65 C, using 0.5 mm EF-G or EF-G-2 for the intrinsic GTPase assays and 0.05 mm EF-G or EF-G for the ribosome-dependent GTPase assays. Poly(U)-Dependent Poly(Phe) Synthesis Assay The time course of Poly(Phe) synthesis was performed at 60 C using a reaction mixture containing of mm [ 14 C]-Phe-tRNA, 1 mm EF-Tu, 0.25 mm EF-Ts, mm 70S ribosome, 0.01 mg/ml PK, and 0.25 mm EF-G or EF-G-2 in the 50 mm Tris-HCl buffer containing 10 mm MgCl 2, 100 mm NH 4 Cl, 1 mm DTT, 0.1 mm spermidine, 5 mm spermine, 1.25 mm GTP, 2.5 mm phosphoenolpyruvate, and mg/ml poly(u). The [ 14 C]-Phe-tRNA, EF-Tu, EF-Ts, and EF-G were purified as described in the Supplemental Experimental Procedures. The synthesized poly(phe) was precipitated by addition of 5% TCA followed by a 10 min incubation at 95 C. This mixture was poured on a cellulose membrane and washed, and the radioactivity was assayed by using a liquid scintillation counter. Supplemental Data Supplemental Data include Supplemental Results and Discussion, Supplemental Experimental Procedures, Supplemental References, two tables, eight figures, and two movies and can be found with this article online at 751/DC1/. ACKNOWLEDGMENTS We would like to thank Drs. K. Wilson and N. Polacek for helpful discussions. The present work was supported by grants from the Volkswagen Stiftung (to C.M.T.S.), by the European Union (to C.M.T.S.), and by the Senatsverwaltung für Wissenschaft, Forschung, und Kultur Berlin (UltraStrukturNetwerk and Anwenderzentrum). S.R.C. was supported by a grant from the Alexander von Humboldt Stiftung. We thank N. Kamiya, H. Naitow, Y. Kawano, T. Hikima, H. Nakajima, T. Matsu, and M. Yamamoto for their assistance in the data collection at BL45XP and BL26B1 in SPring-8; M. Kawazoe, A. Tatsuguchi, and R. Ushikoshi for sample preparation; and S. Sekine and T. Kaminishi for technical assistance. This work was supported by the RIKEN Structural Genomics/Proteomics Initiative (RSGI), the National Project on Protein Structural and Functional Analysis, Ministry of Education, Culture, Sports, Science, and Technology of Japan (to S.Y.). Received: September 22, 2006 Revised: December 24, 2006 Accepted: January 23, 2007 Published: March 8, 2007 REFERENCES Abel, K., Yoder, M.D., Hilgenfeld, R., and Jurnak, F. (1996). An alpha to beta conformational switch in EF-Tu. Structure 4, Ævarsson, A., Brazhnikov, E., Garber, M., Zheltonosova, J., Chirgadze, Y., al-karadaghi, S., Svensson, L.A., and Liljas, A. (1994). Three-dimensional structure of the ribosomal translocase: elongation factor G from Thermus thermophilus. EMBO J. 13, Agrawal, R.K., Heagle, A.B., Penczek, P., Grassucci, R.A., and Frank, J. (1999). EF-G-dependent GTP hydrolysis induces translocation accompanied by large conformational changes in the 70S ribosome. Nat. Struct. Biol. 6, al-karadaghi, S., Aevarsson, A., Garber, M., Zheltonosova, J., and Liljas, A. (1996). The structure of elongation factor G in complex with GDP: conformational flexibility and nucleotide exchange. Structure 4, Blanchard, S.C., Kim, H.D., Gonzalez, R.L., Jr., Puglisi, J.D., and Chu, S. (2004). trna dynamics on the ribosome during translation. Proc. Natl. Acad. Sci. USA 101, Blank, J., Grillenbeck, N.W., Kreutzer, R., and Sprinzl, M. (1995). Overexpression and purification of Thermus thermophilus elongation factors G, Tu, and Ts from Escherichia coli. Protein Expr. Purif. 6, Bourne, H.R., Sanders, D.A., and McCormick, F. (1991). The GTPase superfamily: conserved structure and molecular mechanism. Nature 349, Chan, Y.L., Correll, C.C., and Wool, I.G. (2004). The location and the significance of a cross-link between the sarcin/ricin domain of ribosomal RNA and the elongation factor-g. J. Mol. Biol. 337, Cukras, A.R., Southworth, D.R., Brunelle, J.L., Culver, G.M., and Green, R. (2003). Ribosomal proteins S12 and S13 function as control elements for translocation of the mrna:trna complex. Mol. Cell 12, Czworkowski, J., and Moore, P.B. (1997). The conformational properties of elongation factor G and the mechanism of translocation. Biochemistry 36, Czworkowski, J., Wang, J., Steitz, T.A., and Moore, P.B. (1994). The crystal structure of elongation factor G complexed with GDP, at 2.7 Å resolution. EMBO J. 13, Dasmahapatra, B., and Chakraburtty, K. (1981). Protein synthesis in yeast. I. Purification and properties of elongation factor 3 from Saccharomyces cerevisiae. J. Biol. Chem. 256, Datta, P.P., Sharma, M.R., Qi, L., Frank, J., and Agrawal, R.K. (2005). Interaction of the G 0 domain of elongation factor G and the C-terminal domain of ribosomal protein L7/L12 during translocation as revealed by cryo-em. Mol. Cell 20, Frank, J., and Agrawal, R.K. (2000). A ratchet-like inter-subunit reorganization of the ribosome during translocation. Nature 406, Frank, J., Radermacher, M., Penczek, P., Zhu, J., Li, Y., Ladjadj, M., and Leith, A. (1996). SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, Gavrilova, L.P., Koteliansky, V.E., and Spirin, A.S. (1974). Ribosomal protein S12 and non-enzymatic translocation. FEBS Lett. 45, Hansson, S., Singh, R., Gudkov, A.T., Liljas, A., and Logan, D.T. (2005a). Crystal structure of a mutant elongation factor G trapped with a GTP analogue. FEBS Lett. 579, Hansson, S., Singh, R., Gudkov, A.T., Liljas, A., and Logan, D.T. (2005b). Structural insights into fusidic acid resistance and sensitivity in EF-G. J. Mol. Biol. 348, Hilgenfeld, R. (1995). Regulatory GTPases. Curr. Opin. Struct. Biol. 5, Hwang, Y.W., McCabe, P.G., Innis, M.A., and Miller, D.L. (1989). Sitedirected mutagenesis of the GDP binding domain of bacterial elongation factor Tu. Arch. Biochem. Biophys. 274, Molecular Cell 25, , March 9, 2007 ª2007 Elsevier Inc. 763