Figure S3. Details regarding rec-γ-TuRC... | Rockefeller University Press

Figure S3.

$Details regarding rec-γ-TuRC consensus reconstruction and model building. (A) Schematic of the γ-TuRC highlighting subunit composition and numbering across the complex. (B) Top: two views of the rec-γ-TuRC consensus map showing higher resolution features. Map was sharpened in CryoSPARC and postprocessed with EMready (He et al., 2023). Bottom: two views of the refined rec-γ-TuRC model, including the NEDD1 pinwheel. (C) Two views of the NEDD1 pinwheel predicted by AlphaFold 3 (cartoon representation) fitted into the pinwheel density in the rec-γ-TuRC consensus map (transparent surface). (D) Cartoon representation view of MZT1:GCP5-NHD at the rec-γ-TuRC seam with the consensus density map in transparent surface representation. GCP5 K100 and γ-tubulin K410, identified as cross-linked residues in the native human γ-TuRC, are indicated (Consolati et al., 2020). (E) Left: western blot of inputs and bound fractions of SBP pulldowns of GCP-SBP-Myc constructs from HEK293T cells. GCP6mut corresponds to a deletion of GCP6 residues 329–341, while GCP5mut corresponds to a quadruple mutant of GCP5 R213A/R228G/L256E/V258E. Cells untransfected with any GCP-SBP-Myc constructs served as a negative control. Black triangle indicates location where blots were cropped for final figure generation. The experiment was performed three times with similar results. Right: partial alignment error plot for the AlphaFold prediction in Fig. 2 I. (F) Cartoon representation of AlphaFold 3 prediction of three copies each of GCP2 and GCP3 GRIP1 domains, together with the GCP6 belt and residues 191–252, colored by pLDDT (left) and subunit (right). (G) Segmented surface representation of the previously described helical element lining the lumenal face of GCP6, 2, and 3 in EMDB-11888 (Zimmermann et al., 2020). Map was postprocessed with EMready (He et al., 2023). The γ-TuRC subunits from the same study are shown in cartoon representation for reference. A zoomed in view of an unassigned helix contacting GCP6 is shown on the right and at a higher threshold. (G) Cartoon representation of AlphaFold 3 prediction of GRIP1 domains of GCP4, GCP5 (including NHD), GCP6, and GCP2, as well as MZT1 and the GRIP1 and GRIP2 domains of GCP3, colored by pLDDT (left) and subunit (right). The GCP5 insertion element that contacts the lumenal face of GCP6 is indicated. (H and I) Segmented surface representation of the previously described helical element lining the lumenal face of GCP6, 2, and 3 in EMDB-11888 (Zimmermann et al., 2020). Map was postprocessed with EMready (He et al., 2023). The γ-TuRC subunits from the same study are shown in cartoon representation for reference. A zoomed in view of an unassigned helix contacting GCP6 is shown in I (left) and at a higher threshold. The right shows the GCP5 insertion (aa 567–608) modeled in this study in cartoon representation and fitted into the EMDB-11888 density map (Zimmermann et al., 2020). (J) The GCP5 insertion modeled in this study (aa 567–608) is shown in cartoon representation in the rec-γ-TuRC density map. γ-TuRC position 12 corresponding to GCP6 is indicated in panels G, I, and J for reference. pLDDT, predicted local distance difference test. Source data are available for this figure: SourceData FS3.$

Details regarding rec-γ-TuRC consensus reconstruction and model building. (A) Schematic of the γ-TuRC highlighting subunit composition and numbering across the complex. (B) Top: two views of the rec-γ-TuRC consensus map showing higher resolution features. Map was sharpened in CryoSPARC and postprocessed with EMready (He et al., 2023). Bottom: two views of the refined rec-γ-TuRC model, including the NEDD1 pinwheel. (C) Two views of the NEDD1 pinwheel predicted by AlphaFold 3 (cartoon representation) fitted into the pinwheel density in the rec-γ-TuRC consensus map (transparent surface). (D) Cartoon representation view of MZT1:GCP5-NHD at the rec-γ-TuRC seam with the consensus density map in transparent surface representation. GCP5 K100 and γ-tubulin K410, identified as cross-linked residues in the native human γ-TuRC, are indicated (Consolati et al., 2020). (E) Left: western blot of inputs and bound fractions of SBP pulldowns of GCP-SBP-Myc constructs from HEK293T cells. GCP6mut corresponds to a deletion of GCP6 residues 329–341, while GCP5mut corresponds to a quadruple mutant of GCP5 R213A/R228G/L256E/V258E. Cells untransfected with any GCP-SBP-Myc constructs served as a negative control. Black triangle indicates location where blots were cropped for final figure generation. The experiment was performed three times with similar results. Right: partial alignment error plot for the AlphaFold prediction in Fig. 2 I. (F) Cartoon representation of AlphaFold 3 prediction of three copies each of GCP2 and GCP3 GRIP1 domains, together with the GCP6 belt and residues 191–252, colored by pLDDT (left) and subunit (right). (G) Segmented surface representation of the previously described helical element lining the lumenal face of GCP6, 2, and 3 in EMDB-11888 (Zimmermann et al., 2020). Map was postprocessed with EMready (He et al., 2023). The γ-TuRC subunits from the same study are shown in cartoon representation for reference. A zoomed in view of an unassigned helix contacting GCP6 is shown on the right and at a higher threshold. (G) Cartoon representation of AlphaFold 3 prediction of GRIP1 domains of GCP4, GCP5 (including NHD), GCP6, and GCP2, as well as MZT1 and the GRIP1 and GRIP2 domains of GCP3, colored by pLDDT (left) and subunit (right). The GCP5 insertion element that contacts the lumenal face of GCP6 is indicated. (H and I) Segmented surface representation of the previously described helical element lining the lumenal face of GCP6, 2, and 3 in EMDB-11888 (Zimmermann et al., 2020). Map was postprocessed with EMready (He et al., 2023). The γ-TuRC subunits from the same study are shown in cartoon representation for reference. A zoomed in view of an unassigned helix contacting GCP6 is shown in I (left) and at a higher threshold. The right shows the GCP5 insertion (aa 567–608) modeled in this study in cartoon representation and fitted into the EMDB-11888 density map (Zimmermann et al., 2020). (J) The GCP5 insertion modeled in this study (aa 567–608) is shown in cartoon representation in the rec-γ-TuRC density map. γ-TuRC position 12 corresponding to GCP6 is indicated in panels G, I, and J for reference. pLDDT, predicted local distance difference test. Source data are available for this figure: SourceData FS3.

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