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 Angstrom resolution by X-ray crystallography. A synthetic mercury-containing esterase inhibitor and rece ptor analogue, 9-acetamidosialic acid alpha-thiomethylmercuryglycoside, was designed as the single isomorphous heavy-atom derivative. The asymmetric u nit of one crystal form (form I; P4(3)22, a = b = 155.4, c = 414.4 Angstrom ) contained an HEF trimer. Six mercury sites identifying the three haemaggl utination and three esterase sites were located by difference Patterson map analysis of a 6.5 Angstrom resolution derivative data set. These positions defined the molecular threefold-symmetry axis of the HEF trimer, A molecul ar envelope was defined by averaging a 7.0 Angstrom resolution electron-den sity map, phased by single isomorphous replacement (SIR), about the non-cry stallographic threefold-symmetry axis. Iterative non-crystallographic symme try averaging in real space, solvent flattening and histogram matching were used to extend the phases to 3.5 Angstrom resolution. Molecular replacemen t of the model into a second crystal form (form II; P4(3)2(1)2, a = b = 217 .4, c = 421.4 Angstrom) containing two HEF trimers per asymmetric unit perm itted iterative ninefold averaging of the electron density. The 3.5 Angstro m electron-density map allowed an unambiguous tracing of the polypeptide ch ain and identification of N-linked carbohydrates. The model has been refine d by least squares to 3.2 Angstrom resolution (R-free = 26.7%).