Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1
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1 Supplementary Figure 1 Cryo-EM structure and model of the C. thermophilum 90S preribosome. a, Gold standard FSC curve showing the average resolution of the 90S preribosome masked and unmasked (left). FSC curves of the model against the overall 3.2 Å map (red), the final model against one of the two half maps versus the same half map (FSC work ; green), and the model against one of the two half map versus the second half map (FSC free; cyan) (middle). The orientation distribution of the projections is shown on right. b, Side view of the cryo-em map of the 90S preribosome colored according to local resolution as calculated by ResMap. c, Two side views of the 90S preribosome model. The WD40 domain of Rrp9(PDB: 4J0X)
2 was used as placeholder for the missing WD40 domains of UTP-A complex, UTP-B complex and Enp2. Utp20 could not be modeled and is shown as original electron density. d, Detail view of protein -helix (left), -sheet (middle) and RNA A-helix (right)
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4 Supplementary Figure 2 Structures of nonribosomal biogenesis factors (part 1). The components of UTP-A, UTP-B, U3 snornp, Mpp10 complex and Bms1 complex are shown as ribbons (upper row). The isolated complete density maps are shown in the same view colored according to the local resolution (middle row), and density snapshots from each biogenesis factor with molecular models are also shown (lower row).
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6 Supplementary Figure 3 Structures of nonribosomal biogenesis factors (part 2). Continued from Supplementary Figure 2, the detailed structures of the remaining individual biogenesis factors. Isolated electron density map and molecular models of the 35S rrna and the U3 snorna are also shown. Boxes show the zoom on three RNA regions displaying h43 from the pre-18s region, H8 from the 5 ETS region and a k-turn from the U3 snorna.
7 Supplementary Figure 4 Interactions in UTP-A and UTP-B. a, Overall structure of the UTP-A complex. Here, the WD40 domain of Rrp9 (PDB: 4J0X) represents the corresponding part of Utp4, Utp17 and the entire Utp8. The UTP-A oligomerization domain is represented by poly-alanine helices. b, Two views highlighting the location of the UTP-A complex in context of the 90S preribosome. c, Close-up view on the detailed interaction between the C-terminus of Utp17 and the N-terminus of Utp10. In agreement with the previously observed resistance of the complex to high salt, the interaction is mediated by hydrophobic residues from both proteins forming a hydrophobic interaction surface. d, Overall structure of the UTP-B complex. Here, four WD40 domains could not be unambiguously assigned, but according to their location, they likely belong to Utp12 and Utp13. e-f, The overall (e) and the close-up view (f) demonstrating the location of UTP-B complex in the context of 90S preribosome. To indicate the location of Utp12 and Utp13, also h44 of the pre-40s is highlighted. g, Close-up view on the interaction of Utp1 (ribbon) with Utp21 (surface).
8 Supplementary Figure 5 The structure of the pre-40s domains. a-c, The overall structure of the pre-40s subunit domains within the 90S particle. Close-up are shown for the 5 domain (a), the platform (b) and part of the head (c) of the pre-40s. All sub-domains of the pre-18s rrna were colored according to Fig. 4. Note that ribosomal proteins (es4, us4, es6, es8, us12 and es24 for 5 Domain; es1, es7, us8, us11, us15 for the platform; us7, us9 and es28 for part of the head (h28-30 and h41-h43)) are already in their mature position and interact with the correct rrna region. They were all fitted into the density. d, Close-up view on part of the 3 Major domain (h31-h34) of 18S rrna as represented by a low resolved electron density (shown in purple). In the proposed model, h31 would be located close to the unambiguously fit h30, and its location would be consistent with the closeby position of Emg1. e, View focusing on the location of the other part of the 3 Major domain (h35-h40). Extra electron density for h35-h40 (purple) is located on top of the Emg1 dimer. Electron density for the 90S preribosome was low-pass filtered to 6 Å and filtered to 12 Å for the 3 Major domain. f, The immature rrna helix h16 is shown in color with mature state in grey. The position of h16 was based on superposition with the 5 Domain. g, h18 (left), h28 and h44 (right) are shown as model with density.
9 Supplementary Figure 6 Overall structure of the U3 snornp. a, Ribbon model of the U3 snornp structure shown in electron density (transparent). In addition, four regions from 35S rrna (Box A and BoxA of pre-18s rrna, 5 - and 3 hinge of 5 ETS) which hybridize with U3 snorna are shown. b, Close-up views on BoxA, BoxA, BoxB/C, 5 Hinge, BoxC /D and 3 Hinge duplex of the U3 snorna shown as sticks models and colored according to a. c, Model of the U3 snorna. d-e, The proteins of the U3 snornp coordinate both UTP-A and UTP-B complex (d) as well as other biogenesis factors like Utp3 and Fcf2 (e).
10 Supplementary Figure 7 Structures of Utp2 and Mpp10 complexes. a, Extra density of Utp2-Noc4 cluster (pink) is located on the top surface of Utp15 and close to the Emg1 dimer. The density shows features characteristic for an α-helical HEAT repeat structure and thus is likely representing Noc4. The remaining triangular shaped density connecting with C-terminal helix of Utp2 and the Emg1 dimer likely represents Utp2. b, Close-up view on the interaction between Imp3 and Mpp10. Two helices of Imp3 form a hydrophobic surface and interact with Mpp10 (residues ). Residues in this interface crucial for interaction are labeled. c, Close-up view on the interaction between Imp4 and Mpp10. The β-sheet of Imp4 forms a wide surface for Mpp10 residues , representing a mixture of hydrophobic and hydrogen bond interactions.
11 Supplementary Figure 8 The sequential binding model for the 90S preribosome assembly. Structural analysis suggests dividing in 7 steps illustrated from left to right: Start with early 5 ETS Helix1-2 and UTP-A complex, via complete 5 ETS, 5 Domain, Central, 3 Major, 3 minor and ITS1.
12 Supplementary Figure 9 Structures of RNA-interacting WD40-domain proteins. All assigned WD40 domain proteins in the 90S preribosome that interact with RNAs shown as ribbons. Basically, they can be divided into three clusters: Utp1-WD1, Utp1-WD2, Utp7, Utp17-WD1, Sof1 and Utp21-WD2 interact with RNA using their side surfaces; Utp4- WD1, Utp18 and Rrp9 interact with RNA using their top surfaces; Utp21-WD1 interact with RNA using its bottom surface. Based on this analysis, the WD40 domain represents an important RNA binder that can interact with RNA using multiple binding surfaces.
13 Supplementary Figure 10 Secondary-structure diagram of mature and immature 18S rrna. The immature 18S rrna as observed in the 90S particle is shown in full scale and only the region around the pseudoknot is shown for mature 18S rrna. Different sub-domains are color coded. The connecting sequences of 5 ETS, U3 snorna and ITS1 are shown by a dashed line.
14 Supplementary Table 1 Protein and RNA in the model Protein/RN A Total residue s Modelled Quality Template (PDB ID) Chain ID Nop Built & R/S Nop * T U3 snornp Nop * U Snu V/W Rrp * X U3 RNA * 2 UTP-A complex Utp * Built & D Utp G Utp * L Utp * M Utp1/Pwp * Built & Utp * X-ray structure docked & 5IC8, extension s de novo A E UTP-B complex Utp X-ray 5ICA I Utp * structure docked J Utp * X-ray structure docked & Utp * Built & 5IC7, extension s de novo N O Mpp10 complex Imp Built & a Imp * b
15 Supplementary Table 1 Protein and RNA in the model Mpp / c Bms1 complex Rcl * Homology model docked Bms * Built & 4CLQ Y Z Utp Built & B Utp Built & C Utp * Built & F Utp * Built & H Utp / Built & K Other factors Utp Built & Utp * Homology model docked 1CJS P Q Sof Built & d Emg * Homology model docked 3OIJ e/f Krr Built & g Dim2/Pno * Homology model docked 3AEV h
16 Supplementary Table 1 Protein and RNA in the model Kre * Homology model docked Fcf Built & Faf Built & 35S RNA * Built & 2ZPA i/j k 0 1 Ribosome Ribosomal Protein us4, us7, us9, us12, es28 Built & l-z es1, es4, es6, es7, es8, us11, us15, us17, us18, us24 Homology models docked 4V5O * Model contains gaps
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