GraDeR: Membrane Protein Complex Preparation for Single-Particle Cryo-EM.
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.
3D Electron Cryomicroscopy Group, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Florian
F
Hauer
Department of Life Science, Picobiology Institute, Graduate School of Life Science, University of Hyogo, 3-2-1 Kouto, Kamigori, Ako, Hyogo 678-1297, Japan; Japan Science and Technology Agency (JST), Core Research for Evolutional Science and Technology (CREST), Kawaguchi 332-0012, Japan. Electronic address: gerle.christoph@gmail.com.
Christoph
C
Gerle
3D Electron Cryomicroscopy Group, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Niels
N
Fischer
Cellular and Structural Physiology Institute, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan.
Atsunori
A
Oshima
Department of Life Science, Picobiology Institute, Graduate School of Life Science, University of Hyogo, 3-2-1 Kouto, Kamigori, Ako, Hyogo 678-1297, Japan.
Kyoko
K
Shinzawa-Itoh
Department of Life Science, Picobiology Institute, Graduate School of Life Science, University of Hyogo, 3-2-1 Kouto, Kamigori, Ako, Hyogo 678-1297, Japan.
Satoru
S
Shimada
Department of Molecular Biosciences, Kyoto Sangyo University, Kamigamo Motoyama, Kita-ku, Kyoto 603-8555, Japan.
Ken
K
Yokoyama
Cellular and Structural Physiology Institute, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan.
Yoshinori
Y
Fujiyoshi
3D Electron Cryomicroscopy Group, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany; Department of 3D Electron Cryomicroscopy, Institute of Microbiology and Genetics, Georg-August Universität, 37077 Göttingen, Germany. Electronic address: hstark1@gwdg.de.
Holger
H
Stark
D000818:Animals; D002499:Centrifugation, Density Gradient / Q000379:methods; D020285:Cryoelectron Microscopy / Q000379:methods*; D005990:Glycerol / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D046912:Multiprotein Complexes / Q000737:chemistry / Q000302:isolation & purification
D008565:Membrane Proteins; D046912:Multiprotein Complexes; D005990:Glycerol