X-ray crystallographic determination of the ­structure of the influenza C virus haemagglutinin-esterase-fusion glycoprotein

The structure of the haemagglutinin-esterase-fusion (HEF) glycoprotein from influenza C virus has been determined to 3.2 Å resolution by X-ray crystallography. A synthetic mercury-containing esterase inhibitor and receptor analogue, 9-acetamidosialic acid α-thiomethylmercuryglycoside, was designed as the single isomorphous heavy-atom derivative. The asymmetric unit of one crystal form (form I; P4₃22, a = b = 155.4, c = 414.4 Å) contained an HEF trimer. Six mercury sites identifying the three haemagglutination and three esterase sites were located by difference Patterson map analysis of a 6.5 Å resolution derivative data set. These positions defined the molecular threefold-symmetry axis of the HEF trimer. A molecular envelope was defined by averaging a 7.0 Å resolution electron-density map, phased by single isomorphous replacement (SIR), about the non-crystallographic threefold-symmetry axis. Iterative non-crystallographic symmetry averaging in real space, solvent flattening and histogram matching were used to extend the phases to 3.5 Å resolution. Molecular replacement of the model into a second crystal form (form II; P4₃2₁2, a = b = 217.4, c = 421.4 Å) containing two HEF trimers per asymmetric unit permitted iterative ninefold averaging of the electron density. The 3.5 Å electron-density map allowed an unambiguous tracing of the polypeptide chain and identification of N-linked carbohydrates. The model has been refined by least squares to 3.2 Å resolution (R_free = 26.7%).