3-DIMENSIONAL RECONSTRUCTION OF ANDROCTONUS-AUSTRALIS HEMOCYANIN LABELED WITH A MONOCLONAL FAB FRAGMENT

An immunocomplex formed by the 4 x 6 meric hemocyanin of the scorpion Androctonus australis with the monoclonal antibody L104 was studied by cryoelectron microscopy and subjected to three-dimensional (3D) recon struction. The reconstructed particle reflects the structure of the im munocomplex in its hydrated state and is devoid of the flattening that was previously observed with a negatively stained preparation. A 3D f itting of the X-ray data of the Panulirus interruptus hemocyanin and o f the Fab R19.9 to the reconstruction volume allowed the first quantit ative measurement of the structural parameters of the antigen, and the localization of the epitope at the surface of subunit Aa6. The indepe ndent alignment of the Fabs and the hexamers provides a direct verific ation for the accuracy of the fit and allows the building of the most detailed model of a cheliceratan 4 x 6meric complex and its attached m onoclonal antibodies. (C) 1995 Academic Press, Inc.