Fig. 2: Reconstructed volume of LARGE1dTM glycosyltransferase.
From: LARGE1 processively polymerizes length-controlled matriglycan on prodystroglycan
a LARGE protein constructs, LARGE1dTM and LARGE2dTM (right panel), used for structural and biochemical assays carry an N-terminal FLAGx3 tag, C-terminal hexahistidine tag, and lack the transmembrane domain. b Two molecules of LARGE1 (grey cartoon) were refined into the 3.7-Å map of LARGE1dTM; protomers are displayed as cyan and purple transparent surfaces. c Orthogonal active sites each coordinating a manganese ion (magenta spheres) on alternate protomers face the same direction. A groove (highlighted surface) connects the xylose (cyan shading) and glucuronate (pink shading) transferase domains. The quaternary structure implies that matriglycan is polymerized using orthogonal active sites on alternate protomers of LARGE1 (yellow double-headed arrow). d Magnified view of a glucuronic acid transferase active site showing residues (sticks, labelled in blue text) that coordinate manganese (violet sphere) and UDP-glucuronate (sticks labelled “Ura” and “GlcA”) in the LARGE1dTM-prodystroglycan (DAG128-340) reconstruction. Coordination distances (magenta dotted lines, Å) are in black text. Other residues that compose the active site are shown as lines with grey labels. e Titration of UDP-GlcA into NHS-red labeled LARGE1dTM alone (open circles) or saturated with prodystroglycan (DAG128-749, closed squares) observed by microscale thermophoresis. Data points and error bars represent the averages and standard error for n = 3 replicates, respectively. Similar experimental results were obtained independently four times. Source data are provided as a Source Data file. f Partial absence of density in the xylose transferase domain (red circle) when reconstructed with no symmetry imposed (purple, C1) aligned with a volume refined with axial symmetry (transparent cyan, C2).
