Insect-borne flaviviruses are the causative agents of several human diseases including dengue fever, West Nile fever, Kunjin virus disease, Japanese encephalitis, tick-borne encephalitis, and yellow fever. The viral protein NS1 is an enigmatic virulence factor that exists first as a membrane-associated dimer in the host ER lumen where it is essential to replication of the positive-sense RNA genome and subsequently as a secreted hexameric proteolipid particle where it interacts with proteins of the innate immune system. Challenges to production of recombinant, glycosylated, disulfide-linked NS1 were overcome and crystal structures were determined by “S-SAD” from the anomalous scattering of cystine and methionine sulfur atoms. The unusual structure has a hydrophobic face for membrane association and lipid binding and a glycosylated polar face for interaction with the cell surface and immune system. The crystallographic success with moderate-quality NS1 crystals demonstrates that S-SAD is applicable to a broad range of problems in structural biology. Moreover, a strong synergy with new X-ray sources and detectors indicates that it may become a routine method to solve new crystal structures.