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- EMDB-2982: Sub-tomogram average of a mammalian F-type ATP synthase monomer -

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Basic information

Entry
Database: EMDB / ID: EMD-2982
TitleSub-tomogram average of a mammalian F-type ATP synthase monomer
Map dataSub-tomogram average of bovine F-type ATP synthase in a 2D crystal
Sample
  • Sample: 2D crystal of bovine F-type ATP synthase monomers
  • Protein or peptide: F-type ATP synthase
KeywordsF-type ATP synthase / Mitochondria Bovine heart
Biological speciesBos taurus (cattle)
Methodsubtomogram averaging / cryo EM / negative staining / Resolution: 24.0 Å
AuthorsJiko C / Davies KM / Shinzawa-Itoh K / Tani K / Maeda S / Mills DJ / Tsukihara T / Fujiyoshi Y / Kuehlbrandt W / Gerle C
CitationJournal: Elife / Year: 2015
Title: Bovine F1Fo ATP synthase monomers bend the lipid bilayer in 2D membrane crystals.
Authors: Chimari Jiko / Karen M Davies / Kyoko Shinzawa-Itoh / Kazutoshi Tani / Shintaro Maeda / Deryck J Mills / Tomitake Tsukihara / Yoshinori Fujiyoshi / Werner Kühlbrandt / Christoph Gerle /
Abstract: We have used a combination of electron cryo-tomography, subtomogram averaging, and electron crystallographic image processing to analyse the structure of intact bovine F(1)F(o) ATP synthase in 2D ...We have used a combination of electron cryo-tomography, subtomogram averaging, and electron crystallographic image processing to analyse the structure of intact bovine F(1)F(o) ATP synthase in 2D membrane crystals. ATPase assays and mass spectrometry analysis of the 2D crystals confirmed that the enzyme complex was complete and active. The structure of the matrix-exposed region was determined at 24 Å resolution by subtomogram averaging and repositioned into the tomographic volume to reveal the crystal packing. F(1)F(o) ATP synthase complexes are inclined by 16° relative to the crystal plane, resulting in a zigzag topology of the membrane and indicating that monomeric bovine heart F(1)F(o) ATP synthase by itself is sufficient to deform lipid bilayers. This local membrane curvature is likely to be instrumental in the formation of ATP synthase dimers and dimer rows, and thus for the shaping of mitochondrial cristae.
History
DepositionApr 22, 2015-
Header (metadata) releaseApr 29, 2015-
Map releaseApr 29, 2015-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 141.8
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 141.8
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2982.png

Downloads & links

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Map

FileDownload / File: emd_2982.map.gz / Format: CCP4 / Size: 429.7 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSub-tomogram average of bovine F-type ATP synthase in a 2D crystal
Voxel sizeX=Y=Z: 3.3 Å
Density
Contour LevelBy AUTHOR: 141.800000000000011 / Movie #1: 141.8
Minimum - Maximum127.086883540000002 - 156.748321529999998
Average (Standard dev.)138.239059450000013 (±3.54048061)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions394072
Spacing394072
CellA: 132.0 Å / B: 128.7 Å / C: 237.59999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.33.33.3
M x/y/z403972
origin x/y/z0.0000.0000.000
length x/y/z132.000128.700237.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS403972
D min/max/mean127.087156.748138.239

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Supplemental data

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Sample components

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Entire : 2D crystal of bovine F-type ATP synthase monomers

EntireName: 2D crystal of bovine F-type ATP synthase monomers
Components
  • Sample: 2D crystal of bovine F-type ATP synthase monomers
  • Protein or peptide: F-type ATP synthase

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Supramolecule #1000: 2D crystal of bovine F-type ATP synthase monomers

SupramoleculeName: 2D crystal of bovine F-type ATP synthase monomers / type: sample / ID: 1000
Details: 2D Membrane crystals lipids for reconstitution: 1,2-dimyristoyl-sn-glycero-3-phosphocholine
Oligomeric state: 1 / Number unique components: 1
Molecular weightTheoretical: 600 KDa

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Macromolecule #1: F-type ATP synthase

MacromoleculeName: F-type ATP synthase / type: protein_or_peptide / ID: 1 / 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: Mitochondria
Molecular weightTheoretical: 600 KDa

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsubtomogram averaging
Aggregation state2D array

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Sample preparation

Concentration10 mg/mL
BufferpH: 8.2
Details: 40mM Tris-HCL pH8.2 100mM NaCl 0.02%(w/v)NaN3, 0.5mM ADP, 5mM MgCl2, 0.1mM DTT, 0.1mM EDTA
StainingType: NEGATIVE
Details: Plunge frozen in liquid ethane using home-made freezing device
GridDetails: Glow discharged R2/2, 300 copper mesh quantifoil grids
VitrificationCryogen name: ETHANE / Chamber temperature: 100 K / Instrument: HOMEMADE PLUNGER
Method: 2D crystals were mixed 1:1 (v/v) with 6nm collodial gold particles. Three microliters of protein/gold sample was applied to R2/2 300 copper mesh quantifoil grids. Access liquid was removed by ...Method: 2D crystals were mixed 1:1 (v/v) with 6nm collodial gold particles. Three microliters of protein/gold sample was applied to R2/2 300 copper mesh quantifoil grids. Access liquid was removed by blotting for 3s with Whatman #4 paper before plunge-freezing in liquid ethane
Details10mg/ml ATP synthase were mixed with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids) using a lipid to protein ratio of 0.2. Detergent was removed by dialysis using 20 microliter Hampton dialysis buttons and 15kDa membrane cuttoff.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 14942 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 42000
Specialist opticsEnergy filter - Name: quantum / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Sample stageSpecimen holder: Nitrogen cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 °
TemperatureMin: 80 K / Max: 100 K / Average: 90 K
Alignment procedureLegacy - Astigmatism: Objection lend astigmation was corrected on K2 at magnification used for imaging.
Detailstomographic tilt series
DateFeb 1, 2013
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 80 / Average electron dose: 60 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Crystal parametersUnit cell - A: 179.1 Å / Unit cell - B: 171.4 Å / Unit cell - γ: 94.9 ° / Plane group: P 1
CTF correctionDetails: IMOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 24.0 Å / Resolution method: OTHER / Software - Name: IMOD / Number subtomograms used: 2100
DetailsSubtomograms were selected manually and averaged using PEET (Particle Estimation for Electron Tomography, Boulder)

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