Fig. 2. The E2.P_i state structure with bound endogenous cargo.

a 3.4 Å global resolution cryo-EM map, colored as in Fig. 1b. b Cartoon representation, with the cargo helix modeled as a poly-alanine (cyan). c Overview of the surface electrostatics without the modeled helix and with a similar view as Fig. 1e. d Close-views of the cleft and cavity, respectively, including the poly-alanine chain bound to CtSpf1 (cyan) and ScSpf1 (purple). e Cryo-EM density of the captured cargo helix, built as 31 alanines, presumably with the N-terminus in the cytoplasm (note the Christmas tree effect). The close view shows side-chain densities. f Structural comparison of cargo bound to CtSpf1 (E2.P_i^cargo, wheat, cargo in cyan) and ScSpf1 (PDB-ID: 6XMU, orange, cargo in purple) and close-views of the corresponding M-domains. g Residues potentially involved in binding to the cargo helix (all within 5 Å from the modeled polyalanine are shown). Residues highlighted in bold are conserved among P5A-ATPases.