Cross-kingdom structural alignment of TRAPPII-specific subunits. (A and B) AlphaFold predictions (Jumper et al., 2021; Varadi et al., 2022) of Arabidopsis thaliana (At) (A) AtTRS120 and (B) CLUB/AtTRS130 aligned with the open formation of the TRAPPII monomer structure without substrate in Saccharomyces cerevisiae (Sc) resolved with cryo-electron microscopy (in vitro) from Mi et al. (2022). Arabidopsis subunits were mapped onto the ScTRAPPII structure with the align algorithm in PyMOL. Reported root-mean-square deviations (RMSD) were 16.966 Å (3,775 atoms) for AtTRS120/TRAPPC9 and 12.953 Å (1,660 atoms) for CLUB/AtTRS130/TRAPPC10. The ScTRAPPII monomer is depicted in different shades of gray and only the respective Sc subunit where the Arabidopsis subunits aligned with is highlighted in blue-white (ScTRS120 in A and ScTRS130 in B). AtTRS120 and CLUB/AtTRS130 are colored based on their sequence conservation (red: conserved sequences; orange: intermediate conservation; green: plant-specific sequences—as depicted in Fig. 2, A and D). Both Arabidopsis protein structures aligned with their respective yeast subunit. Despite their small protein length differences ScTRS120 (1,289 aa) and AtTRS120 (1,186 aa) share conserved sequences (Cox et al., 2007) and mapped onto each other in the cross-kingdom structural alignment. Note that the β and γ phosphorylation sites of AtTRS120 (magenta sticks) face inwards, toward the active site chamber (including the RAB11/Rab-A GTPase binding pocket) proposed by Mi et al. (2022) and Bagde and Fromme (2022). Related to Fig. S4 and Video 1.