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- EMDB-5319: Three-dimensional structure of bovine respirasome by single parti... -

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Basic information

Entry
Database: EMDB / ID: EMD-5319
TitleThree-dimensional structure of bovine respirasome by single particle cryo-electron tomography
Map dataEM map of respirasome from bovine mitochondria
Sample
  • Sample: Respirasome from bovine mitochondria
  • Protein or peptide: respirasome
KeywordsOxidative phosphorylation / mitochondria
Biological speciesBos taurus (cattle)
Methodsubtomogram averaging / cryo EM / negative staining / Resolution: 22.0 Å
AuthorsDudkina NV / Kudryashev M / Stahlberg H / Boekema EJ
CitationJournal: Proc Natl Acad Sci U S A / Year: 2011
Title: Interaction of complexes I, III, and IV within the bovine respirasome by single particle cryoelectron tomography.
Authors: Natalya V Dudkina / Mikhail Kudryashev / Henning Stahlberg / Egbert J Boekema /
Abstract: The respirasome is a multisubunit supercomplex of the respiratory chain in mitochondria. Here we report the 3D reconstruction of the bovine heart respirasome, composed of dimeric complex III and ...The respirasome is a multisubunit supercomplex of the respiratory chain in mitochondria. Here we report the 3D reconstruction of the bovine heart respirasome, composed of dimeric complex III and single copies of complex I and IV, at about 2.2-nm resolution, determined by cryoelectron tomography and subvolume averaging. Fitting of X-ray structures of single complexes I, III(2), and IV with high fidelity allows interpretation of the model at the level of secondary structures and shows how the individual complexes interact within the respirasome. Surprisingly, the distance between cytochrome c binding sites of complexes III(2) and IV is about 10 nm. Modeling indicates a loose interaction between the three complexes and provides evidence that lipids are gluing them at the interfaces.
History
DepositionJul 5, 2011-
Header (metadata) releaseJul 13, 2011-
Map releaseAug 31, 2011-
UpdateSep 7, 2011-
Current statusSep 7, 2011Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5319_1.tif

Downloads & links

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Map

FileDownload / File: emd_5319.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEM map of respirasome from bovine mitochondria
Voxel sizeX=Y=Z: 3.8 Å
Density
Contour LevelBy AUTHOR: 0.1 / Movie #1: 0.1
Minimum - Maximum-0.230164 - 0.38968
Average (Standard dev.)0.000151277 (±0.0353204)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 486.4 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.83.83.8
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z486.400486.400486.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-62-62-62
NX/NY/NZ125125125
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.2300.3900.000

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Supplemental data

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Sample components

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Entire : Respirasome from bovine mitochondria

EntireName: Respirasome from bovine mitochondria
Components
  • Sample: Respirasome from bovine mitochondria
  • Protein or peptide: respirasome

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Supramolecule #1000: Respirasome from bovine mitochondria

SupramoleculeName: Respirasome from bovine mitochondria / type: sample / ID: 1000
Details: The sample was thawed from storage at -80 degrees Celcius before being loaded onto the grid.
Oligomeric state: One monomer of NADH dehydrogenase binds to one dimer of cytochrome bc1 and one monomer of cytochrome c oxidase
Number unique components: 1
Molecular weightExperimental: 1.7 MDa / Theoretical: 1.7 MDa / Method: blue-native gel electrophoresis

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Macromolecule #1: respirasome

MacromoleculeName: respirasome / type: protein_or_peptide / ID: 1 / Name.synonym: respirasome / Details: Solubilized with digitonin / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (cattle) / Strain: wild type / synonym: bovine / Tissue: heart / Cell: heart muscle cells / Organelle: mitochondrion / Location in cell: inner mitochondrial membrane
Molecular weightExperimental: 1.7 MDa / Theoretical: 1.7 MDa

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 7
Details: 25 mM Tricine, 7.5 mM Bis-Tris, 0.1 mM PMSF, 0.01% digitonin
StainingType: NEGATIVE
Details: 2.5 microliters were mixed with 10-nm gold particles as fiducial markers and applied on glow-discharged 200 mesh Quantifoil support grids with an additional carbon support film. Grids were ...Details: 2.5 microliters were mixed with 10-nm gold particles as fiducial markers and applied on glow-discharged 200 mesh Quantifoil support grids with an additional carbon support film. Grids were blotted at 100% humidity for 4-6 seconds at a blot offset (the longitudinal grid positioning) setting of -3.5, using a Vitrobot Mk3 (FEI).
GridDetails: 200 mesh copper Quantifoil grids with an additional carbon support film
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 89 K / Instrument: FEI VITROBOT MARK III / Details: Vitrification instrument: Vitrobot Mk3 (FEI) / Method: Blot for 4-6 seconds before plunging

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureMin: 15 K / Average: 77 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 34,000 times magnification
Legacy - Electron beam tilt params: 0
Specialist opticsEnergy filter - Name: FEI / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
DateMar 1, 2010
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (2k x 2k) / Digitization - Sampling interval: 0.38 µm / Number real images: 21 / Average electron dose: 80 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 34000
Sample stageSpecimen holder: Multispecimen holder / Specimen holder model: SIDE ENTRY, EUCENTRIC / Tilt series - Axis1 - Min angle: -70 ° / Tilt series - Axis1 - Max angle: 70 °
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsThe particles were selected from the tomograms by cross-correlation to an artificial globular reference followed by manual selection of particle volumes. Average number of projections used in the 3D reconstructions: 2466. Average number of class averages: 1.
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 22.0 Å / Resolution method: OTHER / Software - Name: IMOD, XMIPP, AV3

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Atomic model buiding 1

Initial modelPDB ID:

1bgy

SoftwareName: UCSF Chimera
DetailsProtocol: Rigid Body
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross correlation

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Atomic model buiding 2

Initial modelPDB ID:

1occ

SoftwareName: UCSF Chimera
DetailsProtocol: Rigid Body
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross correlation

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Atomic model buiding 3

Initial modelPDB ID:

3m9s

SoftwareName: UCSF Chimera
DetailsProtocol: Rigid Body
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross correlation

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