Three-dimensional structure of the truncated core of the Saccharomyces cerevisiae pyruvate dehydrogenase complex determined from negative stain and cryoelectron microscopy images

Abstract

Dihydrolipoamide acyltransferase (E2), a catalytic and structural component of the three functional classes of multienzyme complexes that catalyze the oxidative decarboxylation of alpha-keto acids, forms the central core to which the other components are attached. We have imaged by negative stain and cryoelectron microscopy the truncated dihydrolipoamide acetyltransferase core (60 subunits; M(r) = 2.7 x 10(6)) of the Saccharomyces cerevisiae pyruvate dehydrogenase complex. Using icosahedral particle reconstruction techniques, we determined its structure to 25 A resolution. Although the model derived from the negative stain reconstruction was approximately 20% smaller than the model derived from the frozen-hydrated data, when corrected for the effects of the electron microscope contrast transfer functions, the reconstructions showed excellent correspondence. The pentagonal dodecahedron-shaped macromolecule has a maximum diameter, as measured along the 3-fold axis, of approximately 226 A (frozen-hydrated value), and 12 large openings (approximately 63 A in diameter) on the 5-fold axes that lead into a large solvent-accessible cavity (approximately 76-140 A diameter). The 20 vertices consist of cone-shaped trimers, each with a flattened base on the outside of the structure and an apex directed toward the center. The trimers are interconnected by 20 A thick "bridges" on the 2-fold axes. These studies also show that the highest resolution features apparent in the frozen-hydrated reconstruction are revealed in a filtered reconstruction of the stained molecule.

Fig. 1. Fields of stained (A) and frozen-hydrated (B) tE₂

The multiple views of the pentagonal dodecahedron-like structure are apparent. Arrows denote a cross-like projected view of the structure. The field in A has a predominance of the 5-fold view. Magnification bar = 1000 Å.

Fig. 2. tE₂ field with the microscope stage untilted (A) or tilted 32° (B)

Tilt axis is approximately vertical. Five-fold views are seen to interconvert to 2- and 3-fold views upon rotation of the structure as indicated by the numbered arrows. The arrows identify a tE₂ molecule in a thin region of the stain. The oval shape of the molecule in B reveals that it was partially flattened on the grid. Magnification bar = 500 Å.

Fig. 3. Surface-shaded representations of the 5-, 3-, and 2-fold views of the three-dimensional reconstructions from the images of frozen-hydrated and stained tE₂ molecules

The reconstruction of the stained molecules was corrected for the effects of the microscope CTF and filtered by the CTF corresponding to the frozen-hydrated data to effectively treat both sets of data in the same manner (see text). Magnification bar = 100 Å. The lower panel shows a stereo pair of the 3-fold view of tE₂ from the frozen-hydrated reconstruction.

Fig. 4. Surface-shaded representations of frozen-hydrated and stained tE₂ molecules, viewed along a 3-fold symmetry axis and scaled to the same size and corrected (see text)

The “front” and “rear” halves of the reconstructed volumes are shown.

Fig. 5. Serial planes at 5-Å intervals through the reconstructions of the frozen-hydrated and stained tE₂ molecules starting from their outer edge and viewed in the 3-fold direction

The two reconstructions have been scaled to the same size, and the stained data were filtered as described in the text. Three subunits that comprise each vertex of the structure are apparent. The radius at the center of the first panel is 105 Å (frozen-hydrated value).

Fig. 6. Radial density plots of the stained (■) and unstained (⊡) tE₂ molecules

The plots were scaled to normalize the peak densities at ~ 75 and 100 Å. Because of noise artifacts that typically accumulate at the center of three-dimensional reconstructions (31), the data corresponding to 0-25 Å are not shown.

Fig. 7. Two-fold views of tE₂ reconstructions scaled to the same size

A, original stain data; B, CTF-corrected stain data; C, CTF-corrected unstained data; D, original unstained data; E, stain data, CTF-corrected as in B and filtered with the CTF present in the original unstained data (as in D).