a, Distinct lipid binding sites were identified in the structure of nanodisc-reconstituted ALG6, shown in light brown surface representation. Ordered phospholipids and cholesteryl-hemisuccinate molecules are shown in stick representation (green), and the EM density map was contoured at 5 r.m.s.d. and carved to 1.6 Å. b, Electrostatic surface potential of ALG6, with negative charges coloured in red, neutral charges in white, positive charges in blue and bound Dol25-P-Glc depicted in stick representation with carbons in green. The inset shows a zoomed-in view of the kink induced in the dolichol moiety. c, Electrostatic surface potential of ALG6 shown from four angles to indicate potential binding sites for lipid-linked substrates of ALG6 based on the presence of suitable surface cavities. The red solid arrow indicates the binding site of the observed donor substrate Dol25-P-Glc. The red dashed arrows indicate potential binding sites of the acceptor substrate Dol-PP-GlcNAc 2 Man 9 . These sites were chosen based on groove-like features in the transmembrane region that might bind dolichol and positively charged patches at the ER-luminal membrane boundary that could help to bind the pyrophosphate moiety of the acceptor LLO. Binding to these sites would allow the terminal mannose of the A-branch of the acceptor substrate to reach the active site. d, Membrane deformation by ALG6 shown from distinct views. The EM map of the apo-ALG6 complex with the 6AG9-Fab complex in a lipid nanodisc is coloured yellow for ALG6, red (heavy chain) and black (light chain) for 6AG9-Fab, and transparent grey for density indicating the lipid nanodisc. Note that the lipid bilayer of the nanodisc surrounding the ALG6 protein is twisted and thinner in the region opposite the arch formed by EL4.