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- EMDB-21001: Cryo-EM structure of porcine ATP synthase reconstituted in small ... -

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Basic information

Entry
Database: EMDB / ID: EMD-21001
TitleCryo-EM structure of porcine ATP synthase reconstituted in small unilamellar vesicles_I
Map dataCryo-EM structure of porcine ATP synthase reconstituted in small unilamellar vesicles
Sample
  • Complex: Purified ATP synthase reconstituted in liposomes
Biological speciesSus scrofa (pig)
Methodsingle particle reconstruction / cryo EM / Resolution: 19.0 Å
AuthorsMnatsakanyan N / Llaguno MC / Yang Y / Yan Y / Weber J / Sigworth FJ / Jonas EA
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS064967 United States
National Institutes of Health/National Institute on Aging (NIH/NIA)K01AG054734 United States
CitationJournal: Nat Commun / Year: 2019
Title: A mitochondrial megachannel resides in monomeric FF ATP synthase.
Authors: Nelli Mnatsakanyan / Marc C Llaguno / Youshan Yang / Yangyang Yan / Joachim Weber / Fred J Sigworth / Elizabeth A Jonas /
Abstract: Purified mitochondrial ATP synthase has been shown to form Ca-activated, large conductance channel activity similar to that of mitochondrial megachannel (MMC) or mitochondrial permeability transition ...Purified mitochondrial ATP synthase has been shown to form Ca-activated, large conductance channel activity similar to that of mitochondrial megachannel (MMC) or mitochondrial permeability transition pore (mPTP) but the oligomeric state required for channel formation is being debated. We reconstitute purified monomeric ATP synthase from porcine heart mitochondria into small unilamellar vesicles (SUVs) with the lipid composition of mitochondrial inner membrane and analyze its oligomeric state by electron cryomicroscopy. The cryo-EM density map reveals the presence of a single ATP synthase monomer with no density seen for a second molecule tilted at an 86 angle relative to the first. We show that this preparation of SUV-reconstituted ATP synthase monomers, when fused into giant unilamellar vesicles (GUVs), forms voltage-gated and Ca-activated channels with the key features of mPTP. Based on our findings we conclude that the ATP synthase monomer is sufficient, and dimer formation is not required, for mPTP activity.
History
DepositionNov 19, 2019-
Header (metadata) releaseDec 4, 2019-
Map releaseJan 1, 2020-
UpdateJan 1, 2020-
Current statusJan 1, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0225
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0225
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21001.png

Downloads & links

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Map

FileDownload / File: emd_21001.map.gz / Format: CCP4 / Size: 3.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of porcine ATP synthase reconstituted in small unilamellar vesicles
Voxel sizeX=Y=Z: 2.8 Å
Density
Contour LevelBy AUTHOR: 0.0225 / Movie #1: 0.0225
Minimum - Maximum-0.051735077 - 0.08070429
Average (Standard dev.)0.0019711526 (±0.012486588)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions969696
Spacing969696
CellA=B=C: 268.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z969696
origin x/y/z0.0000.0000.000
length x/y/z268.800268.800268.800
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ720720720
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS969696
D min/max/mean-0.0520.0810.002

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Supplemental data

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Mask #1

Fileemd_21001_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM structure of porcine ATP synthase

Fileemd_21001_half_map_1.map
AnnotationCryo-EM structure of porcine ATP synthase
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM structure of porcine ATP synthase

Fileemd_21001_half_map_2.map
AnnotationCryo-EM structure of porcine ATP synthase
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Purified ATP synthase reconstituted in liposomes

EntireName: Purified ATP synthase reconstituted in liposomes
Components
  • Complex: Purified ATP synthase reconstituted in liposomes

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Supramolecule #1: Purified ATP synthase reconstituted in liposomes

SupramoleculeName: Purified ATP synthase reconstituted in liposomes / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Sus scrofa (pig) / Organ: Heart / Organelle: Mitochondria
Molecular weightExperimental: 750 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

Concentration1 mg/mL
BufferpH: 8 / Details: 50 mM Tris-HCl, 150 mM NaCl, 2 mM MgSO4, 1 mM ATP
GridModel: C-flat-1.2/1.3 4C / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV
DetailsPurified ATP synthase reconstituted in liposomes

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 4.219 µm / Calibrated defocus min: 1.871 µm / Calibrated magnification: 47620 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 19.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 7795
FSC plot (resolution estimation)

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