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- EMDB-2676: Electron cryo-microscopy of bovine ComplexI -

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Basic information

Entry
Database: EMDB / ID: EMD-2676
TitleElectron cryo-microscopy of bovine ComplexI
Map dataReconstruction of bovine ComplexI
Sample
  • Sample: NADH:ubiquinone oxidoreductase
  • Protein or peptide: NADH:ubiquinone oxidoreductase
KeywordsNADH dehydrogenase / respiratory complex
Biological speciesBos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.95 Å
AuthorsVinothkumar KR / Zhu J / Hirst J
CitationJournal: Nature / Year: 2014
Title: Architecture of mammalian respiratory complex I.
Authors: Kutti R Vinothkumar / Jiapeng Zhu / Judy Hirst /
Abstract: Complex I (NADH:ubiquinone oxidoreductase) is essential for oxidative phosphorylation in mammalian mitochondria. It couples electron transfer from NADH to ubiquinone with proton translocation across ...Complex I (NADH:ubiquinone oxidoreductase) is essential for oxidative phosphorylation in mammalian mitochondria. It couples electron transfer from NADH to ubiquinone with proton translocation across the energy-transducing inner membrane, providing electrons for respiration and driving ATP synthesis. Mammalian complex I contains 44 different nuclear- and mitochondrial-encoded subunits, with a combined mass of 1 MDa. The 14 conserved 'core' subunits have been structurally defined in the minimal, bacterial complex, but the structures and arrangement of the 30 'supernumerary' subunits are unknown. Here we describe a 5 Å resolution structure of complex I from Bos taurus heart mitochondria, a close relative of the human enzyme, determined by single-particle electron cryo-microscopy. We present the structures of the mammalian core subunits that contain eight iron-sulphur clusters and 60 transmembrane helices, identify 18 supernumerary transmembrane helices, and assign and model 14 supernumerary subunits. Thus, we considerably advance knowledge of the structure of mammalian complex I and the architecture of its supernumerary ensemble around the core domains. Our structure provides insights into the roles of the supernumerary subunits in regulation, assembly and homeostasis, and a basis for understanding the effects of mutations that cause a diverse range of human diseases.
History
DepositionJun 13, 2014-
Header (metadata) releaseJul 9, 2014-
Map releaseSep 17, 2014-
UpdateJul 15, 2015-
Current statusJul 15, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.32
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.32
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4uq8
  • Surface level: 0.32
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_2676.map.gz / Format: CCP4 / Size: 81.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of bovine ComplexI
Voxel sizeX=Y=Z: 1.717 Å
Density
Contour LevelBy AUTHOR: 0.32 / Movie #1: 0.32
Minimum - Maximum-0.69797403 - 2.63536048
Average (Standard dev.)0.00217741 (±0.06465198)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 480.76 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.7171.7171.717
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z480.760480.760480.760
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS280280280
D min/max/mean-0.6982.6350.002

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Supplemental data

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Supplemental map: run1 half1 class001 unfil.map

Filerun1_half1_class001_unfil.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Supplemental map: run1 half2 class001 unfil.map

Filerun1_half2_class001_unfil.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : NADH:ubiquinone oxidoreductase

EntireName: NADH:ubiquinone oxidoreductase
Components
  • Sample: NADH:ubiquinone oxidoreductase
  • Protein or peptide: NADH:ubiquinone oxidoreductase

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Supramolecule #1000: NADH:ubiquinone oxidoreductase

SupramoleculeName: NADH:ubiquinone oxidoreductase / type: sample / ID: 1000
Details: The enzyme was isolated from bovine heart mitochondria in detergent micelles and imaged on ice as single particles
Oligomeric state: 1 / Number unique components: 1
Molecular weightExperimental: 1 MDa / Theoretical: 1 MDa

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Macromolecule #1: NADH:ubiquinone oxidoreductase

MacromoleculeName: NADH:ubiquinone oxidoreductase / type: protein_or_peptide / ID: 1 / Name.synonym: Complex I / Number of copies: 1 / Oligomeric state: 1 / Recombinant expression: No
Source (natural)Organism: Bos taurus (cattle) / synonym: Bovine / Organelle: mitochondria / Location in cell: mitochondrial inner membrane
Molecular weightExperimental: 1 MDa / Theoretical: 1 MDa

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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

Concentration3.5 mg/mL
BufferpH: 8 / Details: 20mM Tris-HCl, 150 mM NaCl, 0.03% Cymal-7
GridDetails: 300 mesh holey-carbon Quantifoil gold grid R 0.6/1 glow discharged in air
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 100 K / Instrument: OTHER
Method: The specimen was vitrified in an environmental plunge-freeze apparatus (Bellare et al, J.Electr. Micros. Tech., 1988, 10, 87-111). Blot for 15-18 seconds after the diameter of the blotted ...Method: The specimen was vitrified in an environmental plunge-freeze apparatus (Bellare et al, J.Electr. Micros. Tech., 1988, 10, 87-111). Blot for 15-18 seconds after the diameter of the blotted meniscus ceases to expand and plunged.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 81495 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 47000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
TemperatureMin: 80 K / Max: 90 K / Average: 85 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 120,000 times magnification once at the start of data collection
DetailsExposure intensity set to give 50 electron/pixel/second at the detector, which translates to ~17 electrons/square_Angstrom/second at the specimen. Each image was exposed for 4 seconds.
DateOct 21, 2013
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Digitization - Sampling interval: 14 µm / Number real images: 1154 / Average electron dose: 64 e/Å2
Details: An in-house built system was used to intercept the frames from the detector at a rate of 18 frames per second.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 4.95 Å / Resolution method: OTHER / Software - Name: Relion
Details: After extraction and ctf estimation of images, frames 1-32 were used for reconstruction, statistical movie processing to compensate for beam-induced movement and b-factor weighting.
Number images used: 25492
DetailsThe particles were manually picked.
FSC plot (resolution estimation)

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