Three-dimensional structure of NADH-dehydrogenase from Neurospora crassa by electron microscopy and conical tilt...

NADH-dehydrogenase (Complex I) is the first complex of the mitochondrial respiratory chain. It is an amphipatic molecule located in the inner mitochondrial membrane and is composed of at least 35 unique subunits encoded by both mitochondrial and nuclear DNA. The whole complex was isolated in detergent from the fungus Neurospora crassa. It is very stable in its isolated form and was analysed as such by electron microscopy. Its mass, determined by dark-field scanning electron microscopy was estimated as 1.12 MDa. The complex was imaged by transmission electron microscopy, by negative staining and by cryo-electron microscopy. A three-dimensional model, with a resolution estimated at 35 A, was calculated from images of negatively stained complexes by the random conical tilt reconstruction technique. This model confirms the general L-shape of the molecule, with arms of equal length and corroborates the hypothesis of a subdivision of the whole complex into three functional domains. Immuno-labelling of the 49 kDA subunit of the peripheral arm allowed its localization within the complex. This is a first step in the subunit mapping of Complex I and the understanding of its activity.