| The mitochondrial respiratory chain complexes NADH: quinone oxidoreductase (Complex I), cytochrome c oxidoreductase (Complex III), cytochrome c oxidase (Complex IV) and ATP synthase are responsible for the production of energy stored as ATP in a process known as oxidative phosphorylation. Blue-native polyacrylamide gel electrophoresis indicated that these single complexes associate into supercomplexes within the inner mitochondrial membrane. Structural characterization of the respiratory chain supercomplexes and their interactions is very important for a better understanding of oxidative phosphorylation and the function of mitochondria in general. Although the high-resolution structures of most of the individual respiratory chain complexes have been solved, we do not have enough information about their association into supramolecular structures. Cryo-electron microscopy (cryo-EM) offers many advantages for the structure determination of large and unstable membrane proteins at medium- and high-resolution, which is one of the great challenges of structural and cell biology. The goal of this proposal is to determine the 3D structures of two mitochondrial supercomplexes, supercomplex I-III2 and dimeric ATP synthase, at a medium resolution by cryo-EM. We will use relatively stable plant supercomplexes purified by Blue-native polyacrylamide gel electrophoresis and subsequent electro-elution. Large sets of cryo-EM projections of single protein molecules, randomly distributed in ice, will be collected and combined into 3D models with EMAN software. We aim to reach a resolution level where we can see how the subunits of the individual complexes interact within the supercomplexes. This will bring us closer to understanding the mechanism of the inner mitochondrial membrane folding and the functional role of the supramolecular organization of oxidative phosphorylation complexes. Key words: mitochondria, cryo-electron microscopy, 3D reconstruction, supercomplex, ATP synthase, Complex I, Cytochrome c oxidoreductase |
