Nsa2 contacts immature rRNA helix 89 in the pre-60S particle. (A) Nsa2 cross-linked rRNA fragments are shown in a 3D model of the 25S pre-rRNA (see also Fig. S5). The identified rRNA cross-link sites are highlighted (H89 in light blue; H42 and H90 in dark blue) as an rRNA ribbon present in the Arx1 pre-60S particle. (B and D) Enlarged view of the cross-linked region of the pre-60S particle with fits of the indicated sequences into assigned (Nsa2 residues 85–96; Nsa2 residues 35–60) and unassigned electron densities (volumes colored in red). rRNA is shown as artificial density (8 Å resolution) except for cross-linked H42, H89, and H90, which are depicted as a ribbon model. B is a view from the subunit-joining side, and C and D are from the solvent side. C shows the complete electron density including Rpl12 (uL11, orange), Rpl5 (uL18, green), and Rsa4 (blue) with bound Nsa2 (85–96) peptide (red). From here, unassigned Nsa2 residues (thin red line) connect to a nearby electron density in which Nsa2_35–60 α-helix is fitted (red). An overview of the complete pre-60S particle including neighbors of the Rsa4–Nsa2 pair is shown in Fig. S5 C.