Fig. 3: Loss of CYB5R3 enzymatic activity after ufmylation.

a Left, crystal structure of human CYB5R3 (PDB 1UMK). FAD and NADH domains are colored yellow and salmon pink, respectively, whereas bound FAD is colored green. Right, close-up view of the domain interface that recognizes FAD. Broken lines indicate possible hydrogen bonds. b Top, Successive HS-AFM images of CYB5R3. Scale, 10 nm; Height, 0–4 nm. Middle, Histograms of the distances between two globular lobes from 194 frames of 1 molecule. Representative images of closed and open conformations with height profiles are shown right. Bottom, Height profiles of simulated AFM images from the crystal structure (PDB ID: 1UMK) (left) and the manually arranged structure (right) with the simulated images. c The closed conformation (left) corresponds to the crystal structure (1UMK) while the open conformation (right) was manually prepared based on the HS-AFM data. CYB5R3 and FAD are shown with surface and space-filling models, respectively. d Color comparison of purified free CYB5R3 with ufmylated CYB5R3. Recombinant CYB5R3 and UFM1-conjugated CYB5R3 were purified in large quantities as described in Fig. S6. e In vitro enzymatic activity of recombinant CYB5R3 (n = 3) and UFM1-conjugated CYB5R3 (n = 3) were measured as described in Methods section. Data are means ± s.e. Statistical analysis was performed by Šidák’s multiple comparison test after one-way ANOVA. f Left panel: CYB5R3^−/− UFSP2^−/− HEK293T expressing indicated constructs were lysed and then subjected to immunoblot analysis with anti-CYB5R3 antibody. Data shown are representative of three separate experiments. Right panel: the reductase activity of microsomal fractions prepared from the aforementioned cells was measured as described in the Methods section. Data are means ± s.e. Statistical analysis was performed by Šidák’s multiple comparison test after one-way ANOVA. g, h The amounts of saturated, monounsaturated, and polyunsaturated phosphatidylcholine and of phosphatidylethanolamine (g) (n = 3) and of cholesterol (h) (n = 4) in CYB5R3^−/− UFSP2^−/− HEK293T expressing UFM1, UFL1, UFBP1, and CYB5R3 or CYB5R3^K214R. Data are means ± s.e. Source data are provided as a Source Data file.