Extended Data Fig. 2: Biochemical characterisation of ScALG3, HsALG9 and GgALG12 Fabs.
From: Structures of ALG3/9/12 reveal the assembly logic of the N-glycan oligomannose core
a-c, Size exclusion chromatography and SDS-PAGE analysis of ScALG3 D71N with Sc3-16 Fab (a), HsALG9 WT with Hs9-8 Fab (b) and GgALG12 E35Q with Gg12-11 Fab (c). In panels a-c, molecular weight markers are indicated in kDa. All samples in panels a-c included the anti-κ-light-chain nanobody. Purification of ScALG3, HsALG9 and GgALG12 were performed multiple times (n > 10) with similar results. Fractions taken for the SDS-PAGE gels are indicated with red lines. d, Multi-point ELISA binding curves for the indicated Fabs. Data (n = 3 biological replicates for Fab each concentration) are presented as mean values ± SD. e, Activity of ScALG3, HsALG9 and GgALG12 in the presence and absence of the Fabs in panels a-c. The reaction products were transferred to a fluorescent peptide (TAMRA-YANATS-NH2) using TbSTT3B and separated by tricine gel electrophoresis. Lanes with glycopeptide cartoons over them serve as glycopeptide standards for the indicated glycan. Activity assays with Fabs were performed at least three times with similar results. f, Sequences of the complementarity determining regions (CDRs) of each Fab used for structural studies. The Kabat numbering system is used for the Fab CDR residues120. g-i, Cryo-EM density maps of g, ScALG3 ternary, h, HsALG9 first ternary, i, GgALG12 ternary and j, HsALG9 second ternary complexes. The cryo-EM density maps are coloured according to the different components as labelled.
