Three-dimensional reconstruction of Limulus polyphemus hemocyanin from cryoelectron microscopy.
Hemocyanin (Hc) the respiratory pigment of the horseshoe crab Limulus polyphemus (Lp) is composed of 48 approximately 75 kDa copper-containing subunits arranged in eight hexameric groups. In this study, we used the random conical tilt series method to do a three-dimensional (3D) reconstruction of Lp Hc observed in vitreous ice. This approach allowed the unambiguous determination of the handedness of the molecule. Lp Hc contains two superimposed 4 x 6mer structures possessing the same structural features as the other 4 x 6meric Hcs, namely flip and flop views and a rocking effect. Moreover, 3D fitting of the X-ray structure of subunit LpII with the reconstruction volume shows that the intra4 x 6meric 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 subunits LpIV. Model building shows that the flop/flop association is the only possible arrangement which allows the assembly of the whole particle. The two alternate constructions (flip/flop and flip/flip) are forbidden because of steric hindrance.
Taveau
J C
JC
Laboratoire des Protéines Complexes, Université de Tours and CNRS EP117, France.
Boisset
N
N
Lamy
J
J
Lambert
O
O
Lamy
J N
JN
D000465:Algorithms; D000818:Animals; D015925:Cryopreservation; D006433:Hemocyanins / Q000648:ultrastructure*; D006737:Horseshoe Crabs; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008854:Microscopy, Electron; D008961:Models, Structural; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013045:Species Specificity
D006433:Hemocyanins