STRUCTURE OF KEYHOLE LIMPET HEMOCYANIN TYPE-1 (KLH1) AT 15-ANGSTROM RESOLUTION BY ELECTRON CRYOMICROSCOPY AND ANGULAR RECONSTITUTION

A three-dimensional reconstruction of keyhole limpet hemocyanin type 1 (KLH1) has been obtained using electron cryomicroscopy at liquid heli um temperatures and single particle image processing. The use of a hig h-contrast embedding medium, 1% (w/v) glucose and 2% (w/v) ammonium mo lybdate (pH 7.0), enables high-resolution electron micrographs to be r ecorded close to focus, i.e. with excellent transfer of high-resolutio n information, while maintaining enough image contrast to localise the individual macromolecules in the images. When low-pass filtered to si milar to 45 Angstrom resolution, the new 15 Angstrom resolution recons truction is very similar to the earlier reconstructions of gastropodan hemocyanins of specimens embedded in vitreous ice. The map shows much detail and reveals many new symmetry elements in this very large cyli ndrical molluscan hemocyanin. The full KLH1 didecamer has D5 pointgrou p symmetry, yet within the KLH1 decameric half-molecules local 2-fold axes have emerged that make the wall of the KLH1 decamer, in spite of its having an exact C5 symmetry only, resemble the D5-symmetric wall o f the decameric cephalopod hemocyanins. In fact, the outside of each t ier of this six-tiered gastropodan hemocyanin was found to have an app roximate D5 symmetry. Local 2-fold axes also relate the ''functional u nits'' within the dimeric ''morphological units'' of the wall and the collar areas of the 8 MDa KLH1 molecule. Certain local-symmetry-relate d surface motifs may be present up to 60 times on the outside wall of this highly symmetric cylindrical hemocyanin. Keyhole Limpet hemocyani n is used clinically as an immunostimulant. The very strong immune rea ction elicited by this hemocyanin may be associated with its intricate hierarchy of local-symmetry components. (C) 1997 Academic Press Limit ed.