Ribulose bisphosphate carboxylase: a two-layered, square-shaped molecule of symmetry 422

Abstract

Electron micrographs and x-ray diffraction patterns of crystals of ribulose bisphosphate carboxylase, probably the most abundant protein on earth, have provided new details of the arrangement of subunits. The eight large subunits and eight small subunits are clustered in two layers, perpendicular to a fourfold axis of symmetry. Viewed down the fourfold axis, the molecule is square-shaped.

Fig. 1

(A) Three-degree kh0 precession photograph, 30-minute exposure. (B) Same as (A), but 4-hour exposure. In (A) the strong, low-order reflections form a pseudo lattice (1/162 Å by 1/162 Å). In (B) the pattern extends to 14.8 Å and reveals the true lattice dimensions (1/230 Å by 1/ 230 Å). The photographs were recorded as described (4). The scale is the same for (A) and (B). (C) Optical diffraction pattern from the circular region in Fig. 2A, with an exposure time of 0.125 second. The low-order reflections form a 1/162-Å square lattice. (D) Same as (C), but with an exposure time of 8 seconds. The true lattice dimensions (1/230 Å by 1/230 Å) are revealed by the presence of weak reflections (indicated by arrows) appearing at centered points in the pseudo lattice.

Fig. 2

(A) Electron micrograph, recorded by methods described (4) of a thin form II RuBPCase crystal revealing a square array of molecules separated by dark strips of stain. The circular area outlined at the lower left was fmiher processed by optical diffraction and filtering. The scale bar represents 100 nm. The packing scheme proposed in the text implies that this micrograph does not accurately portray the distribution of protein (stain excluding material) in the projected structure. If an integral number of unit cells is evenly contrasted by stain, then the “obscured” molecules (C) should appear half as bright as the clear molecules (A) and (B), and the intensity of the 162-Å (1, 1) reflection should be less than the 115-A (2, 0) reflection (Fig. 1D). This is not true in Fig. 1. Other platelet micrographs in which the crystal is contrasted more evenly and optical diffraction patterns show that the 2, 0 type reflections are more intense than the 1, 1 type reflections. Unfortunately these other micro-graphs are confusing because of superposition of additional layers of molecules. (B) The enlarged region shows detail in individual molecules. One unit cell (230 by 230 v̄_s) is outlined. Circles indicate defects in the crystal where presumably single molecules were either lost or excluded during crystal growth. Faint images of molecules do appear in these positions, indicating that the strong molecular images arise from superposition of more than one molecule (9). (C) Same as (B), but the print was underexposed to reveal the additional molecules obscured by the strips of stain (arrows point to two of these). These obscured molecules are squarish, and in projection are positioned among the four clear molecules situated on the 162-Å square lattice. There is little indication for stain excluding material in the region between clear molecules.

Fig. 3

Optical reconstructions from an identical region of the micrograph in Fig. 2: (a) unfiltered reconstruction; (b) enlarged region of the filtered reconstruction; (c) same as (b), but the print is underexposed to show the filtered appearance of “obscured” molecules. Filtering was accomplished by allowing all reflections on the 1/230-Å lattice to pass through holes etched in a copper mask positioned in the transform plane of the diffractometer. The size of the holes (0.2 mm) was selected so as to produce a reconstruction in which one filtered unit cell results from averaging the information contained in approximately 24 molecules. In (b) and (c), the averaged molecules are seen, and the arrow in (b) points to a spike at the corner of a molecule.

Fig. 4

Packing of RuBPCase molecules in crystal form II. Each molecule contains eight large and eight small subunits, arranged in symmetry D₄ (422). Large subunits are represented by large spheres, and small subunits by small spheres. The positions of the small subunits are conjectural. (a) View of crystal parallel to the c-axis. Light molecules correspond to the “clear” molecules of Fig. 2 and shaded molecules to the “obscured” molecules. (b) View of crystal parallel to the a-axis.