Three-dimensional reconstruction of Lumbricus terrestris hemoglobin at 22 angstrom resolution: Intramolecular localization of the globin and linker chains

A 3D reconstruction of the hemoglobin (Hb) of the earthworm Lumbricus terre stris was carried out by the 3D projection alignment method from electron m icroscopy images of a frozen-hydrated specimen at 22 Angstrom resolution. T he results were analyzed by a new approach taking into account the evolutio n of the 210 densities forming the 3D volume as a function of the threshold of surface representation. The whole oligomer with D-6 point-group symmetr y is comprised of 12 hollow globular substructures (HGS) with local 3-fold symmetry tethered to a complex network of linking subunits (linker complex) . The 12 globin subunits of each HGS are distributed around local 3-fold ax is in four layers of three subunits. The first layer, the most external, co ntains monomeric globin chains 2A, 3A, and 5A. The three trimers correspond ing to the nine remaining subunits have one subunit in each of the second ( 2B, 3B, 5B), third (1A, 4A, 6A), and fourth (1B, 4B, 6B) layer. The distanc es between the centers of the globin chains forming the trimers are in the ranges 20-32 Angstrom and 45-52 Angstrom. The linker complex is made up of two types of linking units. The first type forms three loops connecting glo bin chains of the second, third and fourth layers. The average molecular ma ss (Mm) of these subunits was 25 kDa. The second type forms the central str ucture, termed hexagonal toroid, and its 12 connections to the HGS. This st ructure corresponds to a hexamer of a single linking unit with a Mm (31.2 k Da), size and a shape different from those of the HGS loops. A careful stud y of 3D volume architecture shows that each toroid linking unit is bound to the three loops of a HGS pair located in the upper and lower hexagonal lay ers, respectively. As shown in a model of architecture, hexagonal bilayered (HBL) Hbs can be built very simply from 144 globin chains and 42 linker ch ains belonging to two different types. We also propose a simple assembly se quence for the construction of HBL Hbs based on the architecture model. (C) 1999 Academic Press.