Fig. 1: Purification and cryo-EM structure of enzymatically active Mnl1–Pdi1 complex.
a, Scheme of glycan processing of a misfolded glycoprotein during ERAD-L. The conversion of Man8 to Man7 by Mnl1 commits the protein to Hrd1-mediated ERAD. The exposed α1,6-linked mannose is highlighted in purple. b, Scheme showing the rationale of the mannosidase assay. DyLight 800-labeled SBP-tagged CPY* is incubated with the Mnl1–Pdi1 complex and then bound to streptavidin beads. After washing, the beads are incubated with a DyLight 680-labeled fusion of the MRH domain of OS9 and IgM (MRH–IgM). The amounts of CPY* and bound MRH–IgM are determined by SDS–PAGE and fluorescence scanning at two different wavelengths. c, Mannosidase assays were performed in the presence of the indicated components. d, Quantification of experiments shown in c (see ‘Statistics and reproducibility’ in Methods). The means and s.d. of three experiments are shown. e, Cryo-EM density map of the Mnl1–Pdi1 complex. The MHD, Mnl1 loop and CTD of Mnl1 are shown in different colors. In this view, only Trx domain a of Pdi1 is visible (in cyan). f, As in e, but in a view where all Trx domains are visible. g, As in e, but with the model shown in cartoon representation. A Ca2+ ion is bound in the center of the MHD. h, As in f, but with a cartoon model.
