Nature Structural and Molecular Biology: doi: /nsmb Supplementary Figure 1. Experimental approach for enhancement of unbiased Fo Fc maps.

Transcription

1 Supplementary Figure 1 Experimental approach for enhancement of unbiased Fo Fc maps. a, c, Unbiased Fo-Fc maps of the Tth 70S post-catalysis complex at 2.55 Å resolution with (a) or without (c) bulk solvent correction applied to the active site of the ribosome. For clarity, maps are shown only for the terminal A76 residues of the trnas in both A- and Psites. In all three panels, fitted models of a 3 -terminal A76 residue with an attached di-peptide in the A-site or a deacylated A76 residue in the P-site are shown only for reference, as they were not used for refinement and phase calculation. To prevent a specific region of the asymmetric unit from being included into the solvent mask during bulk solvent correction, a sphere of evenly spaced dummy atoms

2 with zero occupancy (b) was used to cover the active site region. Any dummy atom located <2 Å away from any atom of the physical model was excluded. The resulting unbiased F o F c map shown in (c) was calculated using phases derived from exactly the same model as in (a), but without bulk solvent correction applied to the active site.

3 Supplementary Figure 2 Electron density maps for the CPmn and CCPmn preattack complexes. a, c, The chemical structures of CPmn and f-nh-trnai (a), and CCPmn and f-nh-trnai (c). b, d, Unbiased Fo-Fc electron density maps from co-crystallization experiments with the respective substrates: CPmn and f-nh-trnai at 2.9 Å resolution (contoured at +2.0σ) (b), and CCPmn and f-nh-trnai at 2.8 Å resolution (+2.0σ) (d). Bulk solvent correction was not applied to the active site region, as explained in Fig. S1, S2. The color scheme for the trna-ligands is the same as in Fig. 1. A-site bound trna analogs, CPmn and CCPmn, are colored in red.

4 Supplementary Figure 3 Comparison of the Tth 70S and Hma 50S preattack and postcatalysis structures and of the Tth 70S and Hma 50S structures in complex with short trna analogs. Phe a, Superimposed models of the Tth 70S and Hma 50S pre-attack complexes. In the Tth structure, Phe-NH-tRNA is in the A-site and f-nh-trnai is in the P-site, whereas the Hma structure features CChPmn in the A-site and CCApcb in the P-site (PDB code 1VQN). Structural differences between the two models are within experimental error. Hma numbering is shown in brackets in this and subsequent panels. b, Superimposed models of the Tth 70S and Hma 50S post-catalysis complexes. The Tth structure contains fphe Phe-NH-tRNA in the A-site and deacylated trnai in the P-site, while the Hma structure has CCPmn-pcb and CCA in the A- and Psites, respectively (PDB code 1KQS). In the latter structure, several ribosomal residues fail to undergo the movements induced by proper A-site substrate accommodation and residue A2602(A2637) is not held in place by the 3 -termini of the trnas, resulting in a conformation that would clash with the phosphate group of the P-site residue C74. The color guide is shown at the bottom of each panel. c, d, Superimposed models of our Tth 70S pre-attack (c) and post-catalysis (d) complexes and lower resolution Tth 70S complexes from an earlier study (PDB codes 2WDK/2WDL and 2WDG/2WDI for the pre- and post-catalysis structures, respectively). Differences between the new and old models are within experimental error. However, the amount of detail in the structures presented Phe here is greater owing to a nearly 1-Å increase in resolution. e, f, Superimposed models of Tth 70S featuring full-length Phe-NH-tRNA in the A-site with Tth 70S (e) or Hma 50S (f) in complex with the short aminoacyl-trna analogs CPmn and CCPmn. Structural differences between the active sites of the three Tth complexes shown in (e) are within experimental error, while residues U2506, G2583, U2584 and U2585 of the active site of Hma 50S featuring the CPmn analog in the A-site (PDB code 1VQ6) fail to undergo the movements elicited by proper A-site binding (f). Thus, all of the models shown in (e) correspond to the induced state, with a fully accommodated A-site substrate, whereas CPmn and CCPmn complexes of Hma 50S shown in (f) are in uninduced and induced states, respectively. The observed conformation of the active site for each given structure and the color guide are shown at the bottom of each panel. trna backbone and rrna base movements are shown by black arrows. Hydrogen bonds are shown using black dotted lines.

5 Supplementary Figure 4 Schematic diagrams of the hydrogen-bond networks and reaction schemes. Schematic diagrams of the hydrogen-bond networks and reaction schemes are shown for the proton wire (a) and the eight-membered (b) and six-membered (c) proton shuttles. Hydrogen bond distances and the ω angles between three successive non-hydrogen atoms are indicated. The reaction scheme shown in panel (a) is only for the first half of the peptidyl transfer reaction. Nucleophilic attack is shown by red arrows.