Jc. Taveau et al., 3-DIMENSIONAL RECONSTRUCTION OF LIMULUS-POLYPHEMUS HEMOCYANIN FROM CRYOELECTRON MICROSCOPY, Journal of Molecular Biology, 266(5), 1997, pp. 1002-1015
Hemocyanin (Hc) the respiratory pigment of the horseshoe crab Limulus
polyphemus (Lp) is composed of 48 similar to 75 kDa copper-containing
subunits arranged in eight hexameric groups. In this study, we used th
e random conical tilt series method to do a three-dimensional (3D) rec
onstruction of Lp Hc observed in vitreous ice. This approach allowed t
he unambiguous determination of the handedness of the molecule. Lp Hc
contains two superimposed 4 x 6mer structures possessing the same stru
ctural features as the other 4 x 6meric Hcs, namely flip and flop view
s and a rocking effect. Moreover, 3D fitting of the X-ray structure of
subunit LpII with the reconstruction volume shows that the intra4 x 6
meric contacts described in arthropod Hcs also occur within Limulus Hc
. The two half-molecules composing the 8 x 6mer have their flop faces
in contact (flop/flop association), the main links being formed by sub
units LpIV. Model building shows that the flop/flop association is the
only possible arrangement which allows the assembly of the whole part
icle. The two alternate constructions (flip/flop and flip/flip) are fo
rbidden because of steric hindrance. (C) 1997 Academic Press Limited.