Fig. 4.

Avo1 is positioned next to Lst8 in TORC2. a Selected cryo-EM reference-free 2D class averages show a cloud of EM density (highlighted in red) next to the Thumb of TORC2. The additional density is attributed to Avo1 in line with XL-MS and EM localisation experiments²⁴. Below: a slice of the reconstructed TORC2 volume is shown without (left) and with the final map contoured at 3.5 sigma level (right). Extra density attributed to Avo1, which extends the Thumb is highlighted (red circle). b Surface representations of the TORC2 map in a short side view (left) and the palm view (right). Above: TORC2 shown at 3 sigma; below: same views displayed at 7.5 sigma contour level. c The Avo1 CRIM homology model (yellow-green) fits into the additional density near the Tor2 active site. The domain contacts the rim of the Lst8 β propeller (purple) and the FRB-Avo3 interface. The map (transparent grey) is displayed at 4 sigma. The atoms of the active site residues (Asp2279, Asn2284 and Asp2298) are shown as red spheres. d Insertion of GFP into Avo1 after residue 794, immediately C-terminal to the CRIM domain (residues 647–792), generates new density inside the active site cleft of TORC2 in negative stain 2D class averages. New density is highlighted by white stars in the panel above; the active site of TORC2 is marked by white triangles below