(A) Schematic representation of the XBP1u-del-HR1-mu construct used for purification. The construct encodes N-ter (8X-His, 3X-Flag tag and 3C-protease site), hydrophobic region 2 (red), AP (green) and C-ter (HA-tag). Model for nascent chain in the tunnel, and P-site and A-site positions were denoted as well. (B) Transverse section of cryo-EM structure of the paused XBP1u-RNC showing the peptidyl-tRNA (green) with small and large subunits colored in yellow and gray, respectively. Densities for nascent chain, small and large subunit are displayed at contour levels of 1.5, 1.7 and 4.1 σ, respectively. (C) Close-up views showing the two tRNA states of the XBP1u-RNC, post (top panel) and rotated (bottom panel). For the post state (top panel), P- and E-site tRNA are displayed at 3.4 and 3 σ. While small and large subunit densities are shown at 2.7 and 2.8 σ, respectively. For the rotated state (bottom panel), A/P-, P/E-tRNA, large and small subunit densities are shown at 2.6, 3.1, 3.1 and 3.3 σ, respectively. (D) Overview of the XBP1u nascent chain in the ribosomal tunnel. Surface representation of the electron density: nascent chain (green), uL4 (orange), uL22 (blue) and ribosomal tunnel (gray). Densities for nascent chain, large subunit, uL4 and uL22 are displayed at the contour levels of 2.6, 3.9, 3.2 and 4.1 σ, respectively.