Premium
The use of amphipathic polymers for cryo electron microscopy of NADH:ubiquinone oxidoreductase (complex I)
Author(s) -
FLÖTENMEYER MATTHIAS,
WEISS HANNS,
TRIBET CHRISTOPHE,
POPOT JEANLUC,
LEONARD KEVIN
Publication year - 2007
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.2007.01805.x
Subject(s) - cryo electron microscopy , electron microscope , macromolecule , amphiphile , polymer , particle (ecology) , chemistry , membrane , oxidoreductase , crystallography , materials science , biophysics , biochemistry , organic chemistry , copolymer , biology , enzyme , optics , ecology , physics
Summary In the three‐dimensional (3D) structure determination of macromolecules, cryo electron microscopy (cryo‐ EM ) is an important method for obtaining micrographs of unstained specimens for the single‐particle reconstruction approach. For cryo‐ EM , proteins are fixed in a frozen hydrated state by quick‐freezing in a thin water layer on a holey carbon film. Cryo‐ EM of detergent‐solubilized membrane proteins is hindered by the fact that detergents reduce the surface tension of water, so that it is difficult to control the ice thickness and the distribution of protein. Amphipols are a new class of amphipathic polymers designed to handle membrane proteins in aqueous solutions under particularly mild conditions. Amphipol A8‐35 stabilizes NADH :ubiquinone oxidoreductase (complex I) from Neurospora crassa and keeps it water‐soluble in the absence of free detergent. Electron microscope images of quick‐frozen complex I/A8‐35 samples were used for computer‐based single‐particle averaging and 3D reconstruction, and the reconstruction of unstained frozen‐hydrated particles compared with previous detergent‐based reconstructions. The potential of amphipols for cryo‐ EM is discussed.