Increases or decreases in FRET efficiency relative to ribosome-free and nucleotide-free EF-G (ΔE=Ecomplex-Efree) are shown upon GTP binding (a); GDP binding (b), binding to the 70S ribosome containing deacylated tRNAMet in the P site and N-acetyl-Met-Phe-tRNAPhe in the A in the presence of GTP and fusidic acid (c); binding to the 70S ribosome containing deacylated tRNAMet in the P site and N-acetyl-Met-Phe-tRNAPhe in the A site, in the presence of GTP, fusidic acid and viomycin (d). Ensemble FRET measurements were taken for EF-G_301-603(Cy3/Cy5), EF-G_301-541(Cy3/Cy5), and EF-G_401-538(Cy3/Cy5). Error bars show standard deviations for changes in FRET efficiency were calculated from triplicate measurements.