Fig. 3: High-resolution cryo-EM structure determination of the TPP riboswitch using the group IIC intron scaffold.

A Secondary structure of the TPP riboswitch attached to the scaffold through the stem of Domain III (gold) of O.i. Nucleotides in the TPP riboswitch aptamer domain are colored by SHAPE reactivity. Experiments were performed in the presence of ligand; for full SHAPE data, see Fig. S2. The riboswitch is also colored by helix for clarity. On the right, a secondary structure representation of the tertiary structure is shown. A cartoon depiction of thiamine pyrophosphate (magenta) is shown to emphasize the ligand binding pocket. The three magnesium ions observed in the cryo-EM data are shown as green spheres. The Leontis and Westhof nomenclature⁵⁴ is used to denote noncanonical base pairs. B 2D class averages of the scaffold alone and attached to the TPP riboswitch are shown. Signal corresponding to the TPP riboswitch (star) is clearly visible in the 2D classes. C A local resolution map of the scaffold (left) was generated from focused refinement and overlaid on a low threshold globally refined map (white). The locally refined map of the scaffold maintains high-resolution features after the TPP riboswitch is embedded. Density for the TPP riboswitch is visible in the globally refined map at low threshold. A locally refined map of the TPP riboswitch (green) is shown on the right and overlaid on the same low threshold globally refined map (white) for clarity. D A local resolution map for the TPP riboswitch is shown. The resolution surrounding the TPP binding site is 2.5 Å. E A viewing direction distribution plot is shown for the globally refined scaffold TPP data. The scaffold does not show any orientation preference with the added sequence of the riboswitch.