Three-dimensional structure of the truncated core of the Saccharomyces cerevisiae pyruvate dehydrogenase complex determined from negative stain and cryoelectron microscopy images.
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.
D000123:Acetyltransferases / Q000648:ultrastructure*; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D005615:Freezing; D046911:Macromolecular Substances; D008854:Microscopy, Electron / Q000379:methods; D008961:Models, Structural; D011768:Pyruvate Dehydrogenase Complex / Q000648:ultrastructure*; D011994:Recombinant Proteins / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D013194:Staining and Labeling
D046911:Macromolecular Substances; D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D000123:Acetyltransferases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; C494879:LAT1 protein, S cerevisiae