Three-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) reconstruction. The reconstructed particle reflects the structure of the immunocomplex in its hydrated state and is devoid of the flattening that was previously observed with a negatively stained preparation. A 3D fitting of the X-ray data of the Panulirus interruptus hemocyanin and of the Fab R19.9 to the reconstruction volume allowed the first quantitative measurement of the structural parameters of the antigen, and the localization of the epitope at the surface of subunit Aa6. The independent alignment of the Fabs and the hexamers provides a direct verification 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 monoclonal antibodies.