1. Structural and Functional Insights into Human Re-initiation Complexes
Melanie Weisser, Tanja Schäfer, Marc Leibundgut, Daniel Böhringer, Christopher Herbert Stanley Aylett, Nenad Ban 
Molecular Cell 
Volume 67, Issue 3, Pages e7 (August 2017)
DOI: /j.molcel
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2. Molecular Cell 2017 67, 447-456.e7DOI: (10.1016/j.molcel.2017.06.032)
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3. Figure 1 eIF2D and Subunit Joining
(A) Absorption profiles at 260 nm after sucrose density gradient centrifugation under associative conditions of mixed human 40S and 60S subunits (run 1), 40S and 60S in the presence of ΔdII HCV IRES and initiator tRNA (run 2), and 40S, 60S, ΔdII HCV IRES, initiator tRNA, and 6x-His tagged eIF2D (run 3). 80S (peak 1) forms in runs 1 and 2. 60S (peak 2) and 40S (peak 3) remain separated in the presence of eIF2D (run 3).
(B) Binding of eIF2D to the 40S ribosomal subunit was confirmed by a western blot for run 3, using an HRP-conjugated anti-His-tag antibody.
(C) The presence of the ΔdII HCV IRES in complex 2 (run 2) and complex 3 (run 3) was confirmed in a denaturing 7 M urea 10% polyacrylamide gel stained with SYBR Gold.
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4. Figure 2
Overall View over the Human eIF2D and MCT-1/DENR Re-initiation Complexes
(A) Cryo-EM map of the human eIF2D-initiator tRNA-ΔdII HCV IRES-40S complex, low-pass filtered to 8 Å and colored according to the docked structural models: human 40S head and body (gray) (Quade et al., 2015), yeast initiator tRNA (blue) (Basavappa and Sigler, 1991; Hussain et al., 2014), ΔdII HCV IRES (orange) (Quade et al., 2015), MCT-1-like domain (purple) (Tempel et al., 2013), WH domain (pink) (Carlier et al., 2007), and the crystal structure comprising the SUI (green) and SWIB/MDM2 domains (red) (this study).
(B) Domain architecture of eIF2D as observed in (A) with flexible linker regions indicated as dashed lines.
(C) Domain schema for eIF2D, MCT-1, and DENR. Residues marking the borders between domains are shown as numbers.
(D) View from the foot of the 40S (gray model) on the SUI domain (green) and the MCT-1-like domain (purple), both interacting with the initiator tRNA (blue).
(E) View from the E-site onto the MCT-1-like domain (purple). The DUF1947 domain (lower part) interacts with the rRNA; the PUA fold (upper part) binds and stabilizes the tRNA CCA tail (blue).
(F) Close-up of the density for the SUI domain (green) and its N terminus, which forms a hydrophobic three-stranded β sheet (red) with the C terminus of the SWIB/MDM2 fold.
(G) Close-up of the WH domain (pink) interacting with helix h44.
(H) Filtered, unsharpened cryo-EM map of the human MCT-1/DENR-initiator tRNA-ΔdII HCV IRES-40S complex at 10.9 Å with docked models of the 40S head and body, initiator tRNA, and HCV IRES. The map is segmented and colored in analogy to (A). No extra density is visible on helix 44 (black circle).
See also Figures S1–S4 and Table 1.
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5. Figure 3 The SWIB/MDM2-SUI Domain of eIF2D
(A) Crystal structure of the C-terminal half of human eIF2D, revealing how the SWIB/MDM2 and SUI domains are connected by a three-stranded β sheet.
(B) Representative examples of the unbiased initial crystallographic experimental electron density map used for building of the eIF2D SWIB/MDM2-SUI domain from PHENIX.AUTOSOL (Adams et al., 2010) after solvent modification at two contour levels (pink and gray). The refined coordinates of the model are shown as backbone. Two helices within the SWIB/MDM2 subdomain (map contoured at 2 σ (gray) and 3.5 σ (pink) levels) and the hydrophobic β sheet that bridges the SWIB/MDM2 and SUI subdomains (map contoured at 1.8 σ [gray] and 3.5 σ [pink] levels) are shown.
(C) Docking of the SWIB/MDM2 and the SUI domains of the crystal structure into the cryo-EM map reveals only a minor repositioning of both domains relative to each other. Three interaction points between the AUG-bound P-site initiator tRNA and the SWIB/MDM2-SUI domain can be established: viewed from the A-site, SUI loop β1 contacts the codon-anticodon duplex (1); loop β2 binds the D loop at bases C11 and G12 (2); and the SWIB/MDM2 domain and tRNA acceptor stem interact around bases C66, G67, and G68 (3). Map and structural models are shown as in Figure 2, with the EM map corresponding to 40S head and body removed for clarity.
(D) Close-up view of the interaction (1) between the β1 loop of the SUI domain and the codon-anticodon duplex.
(E) Surface representation of the eIF2D SWIB/MDM2-SUI domain colored by electrostatic potential and in complex with the P-site initiator tRNA. Interaction points (1)–(3) show up as positively charged patches (blue). The view is similar to (A).
(F) Superposition of the canonical yeast 48S translation initiation complex (PDB: 3JAP; Llácer et al., 2015) onto the 40S body of this complex. For clarity, only canonical eIF1 and initiator tRNA of the 48S complex are shown (cyan).
See also Figures S3A, S3F–S3H, S5A, S5B, and S6 and Table 2.
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6. Figure 4
Interaction between the eIF2D PUA Domain and the CCA Tail of the Initiator tRNA
(A) Overview of the complex from the E-site. The map and structural models are colored as in Figure 2.
(B) View of the aligned PUA domain of eIF2D (residues 93–182, purple) and of the archaeosine tRNA-guanine transglycosylase (ArgTGT) (residues 506–582, PDB: 1J2B; Ishitani et al., 2003; gray) complexed with a tRNA CCA tail (upper panel). The structurally conserved PUA RNA binding cleft comprises helix α1 and β strands 2 and 6. The lower panel shows a surface representation of the eIF2D PUA domain colored by levels of conservation ranging from low (dark gray) to high (orange), which were determined based on a multiple sequence alignment (Figure S5C). The tentative position of a tRNA-bound methionine residue is indicated with a blue circle (labeled M).
(C) P-site initiator tRNA (blue) as it is bound by the eIF2D PUA domain in this complex, by eIF2γ (PDB: 3JAP; Llácer et al., 2015), and by eIF5B (PDB: 4UJC; Yamamoto et al., 2014).
(D) Structural model of the MCT-1/DENR-initiator tRNA-ΔdII HCV IRES-40S complex. The model was generated by superposition of the MCT-1 crystal structure (Tempel et al., 2013) and a homology model of the DENR SUI domain onto the eIF2D complex. The 40S rRNA is shown in gray, the ΔdII HCV IRES in orange, and yeast initiator tRNA in blue. Phosphorylation site S118 (Nandi et al., 2007) and the missense mutation site P121L described within patients of autism spectrum disorder (Haas et al., 2016) are indicated as spheres. See also Figures S3B, S3E, and S5C.
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7. Figure 5
Comparison between the eIF2D Translation Re-initiation and the Canonical 48S Translation Initiation Complexes
(A) Surface representation of the eIF2D-40S-initiator tRNA complex.
(B) Model of a canonical 48S translation initiation complex based on PDB: 3JAP (Aylett and Ban, 2017; Llácer et al., 2015). Components of translation initiation factors occupying a similar position on the 40S ribosomal subunit (gray) in both complexes are shown in the same color in (A) and (B). Canonical eIF2α and regions of eIF3 that do not appear to have functional counterparts in eIF2D are shown in cyan.
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