1. Volume 25, Issue 5, Pages 751-764 (March 2007)
Structural Basis for Interaction of the Ribosome with the Switch Regions of GTP-Bound Elongation Factors 
Sean R. Connell, Chie Takemoto, Daniel N. Wilson, Hongfei Wang, Kazutaka Murayama, Takaho Terada, Mikako Shirouzu, Maximilian Rost, Martin Schüler, Jan Giesebrecht, Marylena Dabrowski, Thorsten Mielke, Paola Fucini, Shigeyuki Yokoyama, Christian M.T. Spahn 
Molecular Cell 
Volume 25, Issue 5, Pages (March 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1
Cryo-EM Reconstruction of a T. thermophilus 70S•EF-G•GMPPNP 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.
Molecular Cell  , DOI: ( /j.molcel )
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3. 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 γ-phosphate (Pi). The reaction mixture (20 μl) contained 0.5 mM [γ-32P]-GTP. In (A), 0.5 μM 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 μM 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 μl) contains 3.75 pmol [14C]-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−1Σ(x − Σx2/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′ domain highlighted in olive green), blue, cyan, burgundy, and orange, respectively. Switch I is colored red, and helices B1 and B1′ in switch II are yellow. The characteristic helix E1′ between domains I and II is colored magenta. Mg2+ and GTP are shown as a white sphere and as orange sticks, respectively.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 The Ribosome-Bound Structure of EF-G
(A) The result of docking, as rigid bodies, the five domains of EF-G•GDP (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-G•GDP (1FNM; gray) and (C) EF-G-2•GTP (gray) X-ray structures to highlight the similar domain arrangement of rbEFG and EF-G-2.
Molecular Cell  , DOI: ( /j.molcel )
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5. 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  , DOI: ( /j.molcel )
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6. Figure 5
Conformational Changes in Switch I and II Seen by X-Ray Crystallography
(A) A backbone alignment of EF-G-2•GTP (blue) and EF-Tu•GMPPNP•tRNA (1TTT) complex with domain I. EF-Tu and tRNA are colored in gray and orange. Switch I of EF-Tu and the Mg2+ 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 B3 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 3.7–3.8 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 Mg2+ 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 (|Fo| − |Fc| map contoured at 3 σ) corresponding to GTP molecule is shown. Water molecules coordinating the Mg2+ and the γ-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-2•GTP that are important for GTP binding/hydrolysis are shown superimposed on the corresponding regions from EF-Tu•GMPPNP (gray ribbon, 1TTT) and EF-G•GDP (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 Mg2+ and the interacting waters are colored green and red, respectively.
Molecular Cell  , DOI: ( /j.molcel )
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7. 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.
Molecular Cell  , DOI: ( /j.molcel )
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