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- EMDB-3098: Cryo-EM structure of bovine FoF1 ATP synthase -

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Basic information

Entry
Database: EMDB / ID: EMD-3098
TitleCryo-EM structure of bovine FoF1 ATP synthase
Map data3D map of bovine FoF1 ATP synthase
Sample
  • Sample: Bovine mitochondrial FoF1 ATP synthase
  • Protein or peptide: FoF1 ATP synthase
Keywordsmitochondria / bovine heart / Bos taurus / OXPHOS
Biological speciesBos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 11.0 Å
AuthorsHauer F / Gerle C / Fischer N / Oshima A / Shinzawa-Itoh K / Shimada S / Yokoyama K / Fujiyoshi Y / Stark H
CitationJournal: Structure / Year: 2015
Title: GraDeR: Membrane Protein Complex Preparation for Single-Particle Cryo-EM.
Authors: Florian Hauer / Christoph Gerle / Niels Fischer / Atsunori Oshima / Kyoko Shinzawa-Itoh / Satoru Shimada / Ken Yokoyama / Yoshinori Fujiyoshi / Holger Stark /
Abstract: 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 ...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.
History
DepositionJul 15, 2015-
Header (metadata) releaseAug 5, 2015-
Map releaseSep 2, 2015-
UpdateSep 9, 2015-
Current statusSep 9, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 31
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 31
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMDB-3098.png

Downloads & links

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Map

FileDownload / File: emd_3098.map.gz / Format: CCP4 / Size: 39.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D map of bovine FoF1 ATP synthase
Voxel sizeX=Y=Z: 1.6 Å
Density
Contour LevelBy AUTHOR: 31.0 / Movie #1: 31
Minimum - Maximum-83.957611080000007 - 147.609420779999994
Average (Standard dev.)0.00000674 (±10.20802784)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 352.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.61.61.6
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z352.000352.000352.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-800-4
NX/NY/NZ1611358
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-83.958147.6090.000

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Supplemental data

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Sample components

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Entire : Bovine mitochondrial FoF1 ATP synthase

EntireName: Bovine mitochondrial FoF1 ATP synthase
Components
  • Sample: Bovine mitochondrial FoF1 ATP synthase
  • Protein or peptide: FoF1 ATP synthase

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Supramolecule #1000: Bovine mitochondrial FoF1 ATP synthase

SupramoleculeName: Bovine mitochondrial FoF1 ATP synthase / type: sample / ID: 1000
Details: The sample was initially maintained in a buffer containing the detergent lauryl maltose-neopentyl glycol (LMNG). For cryo-EM preparation, free LMNG-monomers and micelles were removed using the GraDeR method.
Oligomeric state: Monomer / Number unique components: 1
Molecular weightExperimental: 600 KDa / Theoretical: 600 KDa

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Macromolecule #1: FoF1 ATP synthase

MacromoleculeName: FoF1 ATP synthase / type: protein_or_peptide / ID: 1 / Name.synonym: F-ATPase, ATP-synthase / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Bos taurus (cattle) / synonym: cattle / Tissue: Heart / Organelle: Mitochondria / Location in cell: Mitochondrial membrane
Molecular weightExperimental: 600 KDa / Theoretical: 600 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
Details: 50 mM HEPES pH 8.0, 100 mM NaCl, 0.1% azide, 0.5 mM ADP and 5 mM MgCl2
GridDetails: 200 mesh copper Quantifoil grids (3.5/3.5) with custom-made holey carbon film covered by custom-made thin continuous carbon film
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: FEI VITROBOT MARK IV
Details: Nitrocellulose paper was used to allow for slow blotting to avoid disintegration of the FoF1 ATP synthase complex
Method: Slow blotting (15 seconds) at low blot force

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 90000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Alignment procedureLegacy - Astigmatism: Using a Cs-corrector from CEOS electron optical aberrations were corrected to residual phase errors of 45degree at scattering angles of >12 to 15 mrad
DateAug 20, 2013
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 3956 / Average electron dose: 50 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: local CTF correction
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 11.0 Å / Resolution method: OTHER / Software - Name: custom-made, IMAGIC-5, Relion, 1.2 / Number images used: 13238

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