Overview of EF-Tu structure bound to the ribosome. (A) Cryo-EM map of the 70S·fMet-tRNA^fMet·Phe-tRNA^Phe·EF-Tu·GDP·kir complex at a resolution of 6.7 Å shown in transparent surface. The atomic model obtained by molecular dynamics flexible fitting (MDFF) is shown in cartoon representation. (B) Atomic model of the ribosome-bound EF-Tu showing a domain orientation most similar to the closed, GTP-bound form. The switch regions are highlighted: switch I (Sw1) in blue, switch II (Sw2) in orange, and P-loop in green. The same coloring scheme is used in all figures. (C) Closeup of the GTPase domain of EF-Tu. The cryo-EM density map is displayed at a lower threshold to make the switch I density visible. Density thresholds are 2.6 σ (A), 2.2 σ (B), and 1.4 σ (C).

Interaction (marked with an asterisk) between His-19 in the P loop of EF-Tu (green) and A2260 in the tetraloop of the SRL (cyan; tetraloop shown in blue). This interaction presumably establishes an anchor point holding one of the wings of the hydrophobic gate (compare Fig. 2) in place. (Inset) The ribosome is shown in the same orientation. The map is contoured at 2.5 σ. This figure is rotated 90° with respect to the view in Fig. 2.

Hydrophobic gate. (A) Schematic representation of the hydrophobic gate formed by EF-Tu residues Ile-60 and Val-20, which prevents His-84 from activating the water molecule and thus catalyzing GTP hydrolysis (G, guanisine). (B) The gate shown in the crystal structure of the ternary complex bound to kirromycin (PDB ID code 1OB2; unpublished data). (C) The ribosome-bound ternary complex model obtained through MDFF displaying conformational changes in the nucleotide binding area. Switch I has moved away, opening the gate on the Ile-60 side; the acceptor stem of the tRNA has moved significantly closer to switch II; and the side chain of His-84 has been repositioned toward the nucleotide, resulting in catalysis of GTP hydrolysis. (D) Open hydrophobic gate seen in the crystal structure of the aurodox-bound EF-Tu [PDB ID code 1HA3 (12)]. The structure suggests that an opening of the hydrophobic gate is necessary for hydrolysis, and exhibits a conformation of the gate very similar to that obtained by cryo-EM and MDFF. A GTP molecule is shown in B–D for comparison purposes, to illustrate the spacial rearrangements in the hydrophobic gating, although the structures depicted were obtained in presence of GDPNP (B) and GDP (C and D). The coordinates of the GTP molecule correspond to those of GDPNP in the crystal structure depicted in B; the coordinates of the nucleotides present in the structures depicted in C and D are very similar.

Structure of switch I in the ribosome-bound EF-Tu. (A) Stereoview illustrating the movement of switch I (blue) toward the junction of helices h8 and h14 of 16S rRNA (yellow). Rigid-body fitting of the crystal structure (green; PDB ID code 1OB2) places switch I close to the SRL (cyan). At low-density threshold, shown as gray mesh, the density corresponding to SRL exhibits a protuberance (marked with an asterisk) around the S turn motif, possibly corresponding to an interaction with switch I (blue) during its movement toward the h8/h14 junction (yellow). The map is contoured at 1.25 σ. (B) Detail of the interaction between switch I and the 16S rRNA. Elements of EF-Tu (red and blue) and the 30S (yellow) and 50S (cyan) subunits are shown along with the crystal structure of switch I (green; PDB ID code 1OB2). (C) Overview of the ternary complex bound to the 30S subunit (yellow). switch I (blue) interacts with the h8/h14 junction; h44 interacts with the latter on one side, and with the decoding center on the other, possibly serving as part of a signal transduction pathway. Landmarks: sp, spur; sh, shoulder; dc, decoding center; h, head.