Structure of Panulirus interruptus hemocyanin at 5 Å resolution

Abstract

The hemocyanin from the spiny lobster Panulirus interruptus, a hexamer with a molecular weight of approximately 540,000, was crystallized in space group P2 1 with two molecules in the unit cell and cell dimensions a = 119.8 A ̊ , b = 193.1 A ̊ , c = 122.2 A ̊ and β = 118.1 ° . With screened precession photographs a three-dimensional set of reflections was collected up to 10 Å resolution. Both the conventional and the fast rotation function programs were applied and gave results that were in excellent agreement with each other. The hemocyanin hexamer has 32 point group symmetry. Its 3-fold molecular axis runs approximately parallel to the crystallographic 2-fold screw axis. X-ray diffraction data to 5 Å resolution were collected by the oscillation method. Rotation function studies with data between 7 and 5 Å resolution confirmed the 10 Å studies and, furthermore, showed that the rotation axes relating subunits within one hexameric molecule can be distinguished from the rotation axes relating subunits belonging to different hexamers in the unit cell. The local 3-fold axis in the hexamer makes an angle of about 6 ° with the crystallographic 2-fold screw axis. For a mercury and a platinum derivative three-dimensional data sets were collected to 5 Å by the oscillation method. The difference Patterson of the platinum derivative could be solved. The eventual number of heavy-atom sites was 36 for the platinum derivative and 70 for the mercury derivative. From the well-occupied sites the point-group symmetry of the molecule could be established accurately. In addition, the centre of the hexamer could be located within 0.2 Å. Protein phases were obtained from isomorphous as well as anomalous differences. A “best” electron density map calculated with these phases showed the shape of the hexameric molecule as well as the boundaries of the six subunits. Correlation coefficients between the densities of the subunits showed little variation, suggesting a random distribution of the different subunit types (Van Eerd & Folkerts, 1981) over the six positions in the hexamer. The subunits are positioned at the corner of an antiprism. When viewed along the 3-fold axis the hexamer is roughly hexagonal in shape, with a diameter of approximately 120 Å. Viewed along one of the 2-fold axes the molecule is of rectangular shape with dimensions 95 Å × 120 Å. The subunit can be described as an ellipsoid of irregular shape with axes of 80 Å, 55 Å and 48 Å. Each subunit makes extensive contacts with three other subunits in the hexamer and, possibly, a much weaker contact with a fourth subunit.