Volume 8, Issue 1, Pages 181-188 (July 2001) The Polypeptide Tunnel System in the Ribosome and Its Gating in Erythromycin Resistance Mutants of L4 and L22  Irene S. Gabashvili, Steven T. Gregory, Mikel Valle, Robert Grassucci, Michael Worbs, Markus C. Wahl, Albert E. Dahlberg, Joachim Frank  Molecular Cell  Volume 8, Issue 1, Pages 181-188 (July 2001) DOI: 10.1016/S1097-2765(01)00293-3

Figure 1 Three-Dimensional Cryo-EM Maps of E. coli Ribosomes Resistant to Erythromycin and Wild-Type Control (A, D, and G) Mutation of L4. (B, E, and H) Mutation of L22. (C, F, and I) Wild-type control (Gabashvili et al., 2000). (A–C) Solvent-side view of the 70S particles. (D–F) Interface view of the 50S subunit portion of the maps. (G–I) Close-ups of the region around the tunnel entrance, the view shown in (D–F). A molecule of erythromycin, oriented to show its bigger dimensions, is also shown for rough size comparison. In (G), the positions of helices h38 and h69 of 23S rRNA, constituting bridges B1a and B2a, are marked. (Note that the zoomed portion in [G–I] does not show the complete opening, as part of it is hidden behind an elevation next to h69). Features marked are as follows: St, L7/L12 stalk; L1, L4, and L22, globular portions of proteins of the large subunit; sp, spur; e1, e2, e3, and e4, exits of the tunnel system; e1 is the primary “polypeptide translocation” exit. Scale bar = 30 Å Molecular Cell 2001 8, 181-188DOI: (10.1016/S1097-2765(01)00293-3)

Figure 4 Tunnel Systems of the 50S Subunit of Mutants and Wild-Type The tunnels are shown in the same views as in Figure 3. (A and B) L4 mutant. (C and D) L22 mutant. (E and F) 70S ribosome used as one of the wild-type controls (Spahn et al., 1999). (G and H) 70S ribosome complexed with EF-G in its GTP state (Agrawal et al., 1999). Arrows mark the site of the tunnel entrance. The red dashed circles in (C) and (D) show the putative site of interior erythromycin binding. The location of section where tunnel entrance width was measured is marked with red arrows. The tunnel systems of the 50S ribosomal subunit depicted here can be viewed in three dimensions at http://www.molecule.org/cgi/content/full/8/1/181/DC1 Molecular Cell 2001 8, 181-188DOI: (10.1016/S1097-2765(01)00293-3)

Figure 2 Initiator tRNA-Complexed E. coli Ribosome 50S Subunit and Tunnel System (A) 50S subunit cut in the plane along the main tunnel, shown with the cutting surface in white, and with the fMet-tRNAfMet masked off the map. (B) Same as (A), with tunnel cast shown in yellow. (C and D) Isolated tunnel system filtered to 17 Å and displayed in the same view, as “positive cast,” (C) for the initiation-like ribosomal complex and (D) for the X-ray structure of the H. marismortui 50S subunit. IC, interface canyon, painted in lighter yellow (here and in Figures 3 and 4) to distinguish it from the connected tunnel system. Other landmarks are labeled as in Figure 1. The location of section where tunnel entrance width was measured is marked with red arrows. Scale bar = 30 Å Molecular Cell 2001 8, 181-188DOI: (10.1016/S1097-2765(01)00293-3)

Figure 3 The H. marismortui Tunnel System and Its Spatial Relationship with Mutation and Protection Sites The tunnel system (yellow) is shown in two distinct orientations (A and B, C) with proteins L4 and L22 (blue); RNA nucleotides are perturbed as a result of mutation in these proteins (red; Gregory and Dahlberg, 1999), erythromycin protection sites (green; Moazed and Noller, 1987), and (C) RNA chains (shown in Ribbons; Carson, 1997) extended from the tunnel to the stalk base region (dark blue, nucleotides 2052–2110, a region connecting domains IV and V; magenta, nucleotides 2479–2577, central loop system of domain V; purple, nucleotides 1128–1234, RNA component of the stalk base). Asterisk (*) marks A2486 (hidden in [A]) of the peptidyl-transferase center (H. marismortui numbering; Ban et al., 2000; Nissen et al., 2000) Molecular Cell 2001 8, 181-188DOI: (10.1016/S1097-2765(01)00293-3)