Fig. 1: Biochemical and cryo-EM characterization of human MICAL1.

a Domain organization of human MICAL1. The four domains, monooxygenase (MO), CH, LIM, and Rab-binding domain (RBD), are interconnected by three loops (L1-L3). Two Rab-binding sites (RBS-I/II) in the RBD are indicated. The cofactor FAD bound in the MO domain is highlighted, which is essential for enzymatic activity. b Analytical size-exclusion chromatographic (aSEC) and SDS-PAGE analyses of the purified MICAL1 sample. The aSEC analysis was coupled with multi-angle static light scattering for molecular weight determination. c Enzymatic activity measurements of the MO fragment and full-length MICAL1. The enzymatic activities were measured using a NADPH-consumption assay. Protein concentration of 1 μM was used. The results were normalized against the activity of the MO fragment. Three independent repeats were performed for each condition. d Representation of two 2D classes of MICAL1 particles, showing the two conformations of MICAL1 observed in cryo-EM analysis. e, f The cryo-EM map (e) and corresponding atomic model (f) of full-length MICAL1 in the autoinhibited state. Each domain is colored using the same code as that in panel (a). The large and small lobes (MO^L and MO^S) in the MO domain and the two helices (H1 and H2) in RBD are labeled. An unassigned piece of density is highlighted transparently in panel (e). The L1-L3 loops missing in the density map are indicated by dash lines, and the N/C-termini of the atomic model of full-length MICAL1 are labeled (f). g Density-fit-model showing the presence of the cofactor FAD in the MO domain. The map counter level is set to 0.1.