| Products/Services Used | Details | Operation |
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| Proteins, Expression, Isolation and Analysis | After membrane solubilization with 1% (w/v) GDN (Anatrace) at 4°C for 2 hours, followed by centrifugation at 20,000g for 1 hour, the supernatant was collected and purified with anti-Flag G1 affinity resin (GenScript). /The resin was washed with buffer A [25 mM Hepes (pH 7.5), 150 mM NaCl, and 0.01% GDN] plus 5 mM adenosine 5′-triphosphate–MgCl2 and eluted with buffer A supplied with Flag peptide (200 μg/ml; GenScript). | Get A Quote |
The serine palmitoyltransferase (SPT) complex catalyzes the first and rate-limiting step in sphingolipid biosynthesis in all eukaryotes. ORM/ORMDL proteins are negative regulators of SPT that respond to cellular sphingolipid levels. However, the molecular basis underlying ORM/ORMDL-dependent homeostatic regulation of SPT is not well understood. We determined the cryo-electron microscopy structure of Arabidopsis SPT-ORM1 complex, composed of LCB1, LCB2a, SPTssa, and ORM1, in an inhibited state. A ceramide molecule is sandwiched between ORM1 and LCB2a in the cytosolic membrane leaflet. Ceramide binding is critical for the ORM1-dependent SPT repression, and dihydroceramides and phytoceramides differentially affect... More
