Three-Dimensional Reconstruction of Native Androctonus Australis Hemocyanin

Hemocyanin, the respiratory pigment of the scorpion Androctonus australis is composed of 24 kidney shaped subunits. A model of architecture supported by many indirect arguments has been deduced from electron microscopy (EM) and immuno-EM. To ascertain, the disposition of the subunits within the oligomer, the 24mer was submitted to three-dimensional reconstruction by the method of single-exposure random-conical tilt series.A sample of native hemocyanin, prepared with the double layer negative staining technique, was observed by transmisson electron microscopy under low-dose conditions. Six 3D-reconstructions were carried out indenpendently from top, side and 45°views. The results are composed of solid-body surface representations, and slices extracted from the reconstruction volume.The main two characters of the molecule previously reported by Van Heel and Frank, were constantly found in the solid-body surface representations. These features are the presence of two different faces called flip and flop and a rocking of the molecule around an axis passing through diagonnally opposed hexamers. Furthermore, in the solid-body surface of the top view reconstruction, the positions and orientations of the bridges connecting the half molecules were found in excellent agreement with those predicted by the model.