GraDeR: Membrane Protein Complex Preparation for Single-Particle Cryo-EM.

Structure (London, England : 1993)

PubMedID: 26278176

Hauer F, Gerle C, Fischer N, Oshima A, Shinzawa-Itoh K, Shimada S, Yokoyama K, Fujiyoshi Y, Stark H. GraDeR: Membrane Protein Complex Preparation for Single-Particle Cryo-EM. Structure. 2015;.
We developed a method, named GraDeR, which substantially improves the preparation of membrane protein complexes for structure determination by single-particle cryo-electron microscopy (cryo-EM). In GraDeR, glycerol gradient centrifugation is used for the mild removal of free detergent monomers and micelles from lauryl maltose-neopentyl glycol detergent stabilized membrane complexes, resulting in monodisperse and stable complexes to which standard processes for water-soluble complexes can be applied. We demonstrate the applicability of the method on three different membrane complexes, including the mammalian FoF1 ATP synthase. For this highly dynamic and fragile rotary motor, we show that GraDeR allows visualizing the asymmetry of the F1 domain, which matches the ground state structure of the isolated domain. Therefore, the present cryo-EM structure of FoF1 ATP synthase provides direct structural evidence for Boyer's binding change mechanism in the context of the intact enzyme.