Volume 25, Issue 12, Pages 1785-1794.e3 (December 2017) The cryo-EM Structure of a Novel 40S Kinetoplastid-Specific Ribosomal Protein  Jailson Brito Querido, Eder Mancera-Martínez, Quentin Vicens, Anthony Bochler, Johana Chicher, Angelita Simonetti, Yaser Hashem  Structure  Volume 25, Issue 12, Pages 1785-1794.e3 (December 2017) DOI: 10.1016/j.str.2017.09.014 Copyright © 2017 Elsevier Ltd Terms and Conditions

Structure 2017 25, 1785-1794.e3DOI: (10.1016/j.str.2017.09.014) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Cryo-EM reconstructions of native and salt-washed 40S complexes from T. cruzi (A) Absorbance profile from 10% to 30% sucrose gradient at 260 nm, taken from the top to the bottom, displaying peaks for 40S (light blue square), 60S, and 80S complexes. (B and C) Cryo-EM reconstruction of native 40S complex (purified in 50 mM KOAc) from T. cruzi viewed from different orientations. Density segments correspond to 40S (yellow) and unassigned densities (red and green). (D) Close-up view on the platform of the 40S, highlighting unassigned density. (E) List of top-hit non-ribosomal proteins detected from native and salt-washed 40S by mass spectrometry. The intensity of the red background indicates the occupancy of the proteins in the sample compared to 40S r-proteins (red = equimolar to 40S r-proteins). NRBD was consistently detected with native and salt-washed (1 M KOAc) 40S complex. (F and G) Cryo-EM reconstruction of salt-washed 40S complex from T. cruzi viewed from solvent and intersubunit sides shows the unassigned density (red) on the 40S left foot. (H) Close-up view on the platform of salt-washed 40S, highlighting the absence of the additional densities at the platform. See also Figures S1–S3 and Table S1. Structure 2017 25, 1785-1794.e3DOI: (10.1016/j.str.2017.09.014) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Conservation of the Uncharacterized Density on the Left Foot of 40S Subunits among Kinetoplastids and Detection of NRBD in the 40S and 80S Ribosome (A) Cryo-EM reconstruction of L. donovani 80S (Zhang et al., 2016), viewed from the SSU side. Density segments correspond to 80S (gray) and the unassigned density located on the foot (red). (B) Close-up view on the L. donovani 40S left foot, highlighting an unassigned density (red) that could be attributed to a protein. (C) Close-up view on the T. cruzi 40S left foot, highlighting the presence of the same density (red) found in L. donovani 80S reconstruction. (D and E) SDS-PAGE gel of r-proteins from fractions of purified T. cruzi 40S subunits and 80S ribosomes. Mass spectrometry analysis of the ≈30 kDa gel band (marked with a red arrow) detected the NRBD protein at equimolar levels when compared with other ≈30 kDa 40S r-proteins. See also Figures S1, S3, and S5. Structure 2017 25, 1785-1794.e3DOI: (10.1016/j.str.2017.09.014) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Fitting of the KSRP Homology Model into Its 80S Cryo-EM Segmented Density (A) View of KSRP bound to the L. donovani 80S ribosome showing two globular domains and a helical C-ter that contact ES3S (orange) and r-protein eS6 (blue). The close-up view (right panel) highlights the interactions of KSRP (red) with ES3S (orange), ES6S (pink), and r-protein eS6 (blue). (B) Atomic model of KSRP (right panel), showing the C-terminal end colored in cyan, RRM1 in green, RRM2 in red, and the α-helical linker colored in yellow. The black dashed line on the left panel represents a most likely flexible 55-aa N-terminal tail (68-aa in TcKSRP), which remained unmodeled due to its lack of density. Structure 2017 25, 1785-1794.e3DOI: (10.1016/j.str.2017.09.014) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Architecture of Ribosome-Bound KSRP (A) L1-stalk side view of the cryo-EM map of T. cruzi 80S, highlighting the 40S (yellow), 60S (cyan), E-tRNA (purple), and KSRP (red). (B) Overview of the atomic model of T. cruzi 80S showing KSRP (red) bound to ES3S (orange ribbon), ES6S (pink ribbon), and r-protein eS6 (dark blue) at the left foot of 40S. (C and D) Close-up views of the electron density (gray mesh) with molecular models for T. cruzi and L. donovani KSRP (red), ES3S (orange), ES6S (pink), and r-protein eS6 (dark blue), highlighting their interaction network within the ribosome and their consistency with the cryo-EM reconstructions. The blow-up squares highlight the main interactions in both Tc and Ld, respectively, of KSRP RRM2 with ES3S (cyan frames), KSRP RRM1 with ES6S (purple frames), and KSRP C-ter with eS6 C-ter, ES3S, and ES6S (black frames). The resolution of L. donovani 80S and to some extent T. cruzi cryo-EM maps (C and D) allowed accurate fitting of most side chains from the KSRP, ES3S, ES6S, and r-protein eS6. See also Figure S4. Structure 2017 25, 1785-1794.e3DOI: (10.1016/j.str.2017.09.014) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 Detailed Ribosomal Interacting Network of KSRP (A) Secondary structure of kinetoplastidian 18S rRNAs. The regions corresponding to ES3S and ES6S are highlighted in bold. The specific KSRP-binding regions are highlighted in orange (ES3S) and pink (ES6S). (B) Alignment of several kinetoplastid 18S rRNA sequences showing the high conservation of KSRP interacting nucleotides within the ES6S (upper panel) and ES3S (bottom panel). The key KSRP interacting residues are shown on the bottom part of the alignment using the same color code as in Figures 4B and S4B. (C) Close-up views highlighting key interactions between KSRP and ES6S and ES3S in the ribosome. See also Figure S4. Structure 2017 25, 1785-1794.e3DOI: (10.1016/j.str.2017.09.014) Copyright © 2017 Elsevier Ltd Terms and Conditions