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. 2021 Oct 26;10:e72353. doi: 10.7554/eLife.72353

Figure 2. Structural basis for selective targeting of SMG1 by the SMG1 inhibitor.

(A) Model of the SMG1-8-9 kinase complex bound to SMG1i. SMG1 is in gray, SMG8 is in blue, and SMG9 is shown in green. SMG1i is shown as a magenta model overlaid with the isolated transparent density. Approximate location of the SMG1 active site is indicated by a black circle. (B) Key interactions of SMG1i with SMG1 active site residues. Important residues located in either the N- or the C-lobe of the SMG1 kinase domain are colored gray. Other parts of SMG1 are transparent and interactions of SMG1i with SMG1 backbone are not shown. (C) Superposition of SMG1i-bound SMG1 with the mTOR active site (PDB identifier: 4JSP) over the catalytic loops of both kinases. Key SMG1 residues indicated in (B) are shown alongside the respective mTOR residues colored in green. Regions possibly accounting for preferential interaction of SMG1i with SMG1 over mTOR are circled and the relevant residues are labeled.

Figure 2.

Figure 2—figure supplement 1. Resolution distribution and isotropy of SMG1-centered cryo-EM maps bound to SMG1i.

Figure 2—figure supplement 1.

SMG1i-bound reconstructions used in this study for model building are colored according to estimated local resolution shown in two different orientations. A three-dimensional Fourier shell correlation (FSC) plot is included for each reconstruction (Tan et al., 2017). The red line represents the estimated global masked half map FSC. The resolutions according to the gold standard FSC cutoff of 0.143 are indicated and shown as a black dashed line (Rosenthal and Henderson, 2003). The spread of directional resolution values is defined as ±1σ (dashed gray lines). Overall isotropy of the maps is indicated by the given sphericity values (out of 1). In the bottom row of each panel, a model versus map FSC is shown alongside a plot visualizing the distribution of particle views. (A) SMG1 body bound to SMG1i after focused refinement (EMD-13676, PDB identifier: 7PW6). (B) SMG1-8-9 bound to SMG1i (EMD-13674, PDB identifier: 7PW4). (C) SMG1-9 bound to SMG1i (EMD-13677, PDB identifier: 7PW7). cryo-EM, cryo-electron microscopy; FSC, XXX.
Figure 2—figure supplement 2. Cryo-EM data processing of SMG1i data set.

Figure 2—figure supplement 2.

(A) 2D class averages of SMG1-8-9 and SMG1-9 are calculated from the final particle stacks. Scale bars≈100 Å. (B) Processing scheme. Processing steps are indicated in blue; particle numbers and percentages with respect to initial candidate particles are shown for relevant classes. Colored, dashed rectangles indicate the different final reconstructions and the respective classes obtained from the data set collected in the presence of SMG1i. cryo-EM, cryo-electron microscopy.
Figure 2—figure supplement 3. Further details of SMG1i binding and specificity.

Figure 2—figure supplement 3.

(A) Multiple sequence alignment of parts of the kinase domains (N- and C-lobe indicated) belonging to the catalytically active members of the PIKK family with residues colored by identity. Residues of special interest are highlighted as indicated. (B) Different views of the isolated cryo-EM density for SMG1i with the fitted model. (C) Close-up side-view of cryo-EM density of SMG1-8-9 active site bound to SMG1i. The inhibitor model is shown and the corresponding segmented density is displayed in transparent magenta. (D) Superposition of SMG1i-bound active site with AMPPNP-bound active site (PDB identifier: 6Z3R). Same view as in (C). (E) Superposition of SMG1i-bound active site with Torin 2-bound mTOR active site (PDB identifier: 4JXP). Similar view as in (C). (F) Superposition of SMG1i-bound SMG1 with DNA-PK active site in the inactive conformation (PDB identifier: 7K11). Figure prepared and labeled as in Figure 2C, with DNA-PK residues colored in red. The asterisk indicates a residue only visible in the DNA-PK structure due to structural rearrangements. (G) Superposition of SMG1i-bound SMG1 with ATR active site (PDB identifier: 5YZ0). Figure prepared and labeled as in Figure 2C, with ATR residues colored in orange. An asterisk indicates corresponding residues in SMG1 and ATR that are separated due to conformational divergence. cryo-EM, cryo-electron microscopy.