||<p>The biosynthesis of many important polysaccharides (including peptidoglycan, lipopolysaccharide,
and N-linked glycans) necessitates membrane transport of oligosaccharide precursors
from their cytoplasmic site of synthesis to their site of assembly outside the cytoplasm.
To address this problem, cells utilize transporters such as those of the multidrug/oligosaccharidyl-lipid/polysaccharide
(MOP) superfamily to flip lipid-linked oligosaccharides across the cytoplasmic membrane.
The MOP superfamily member MurJ has been shown to be the flippase that transports
the lipid-linked peptidoglycan precursor lipid II, but the lack of structural information
has limited our mechanistic understanding of the MurJ transport cycle. We determined
the first crystal structure of MurJ (MurJTA from Thermosipho africanus) to 2.0-Å resolution,
which assumed an inward-facing conformation unlike all other outward-facing structures
of MOP transporters. Our structural and mutagenesis studies provide insight into a
putative model of lipid II binding and an alternating-access mechanism of transport.</p>