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- EMDB-2975: Cryo electron microscopy of yeast vacuolar ATPase proton channel ... -

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Basic information

Entry
Database: EMDB / ID: EMD-2975
TitleCryo electron microscopy of yeast vacuolar ATPase proton channel sector
Map dataReconstruction of yeast V-ATPase membrane sector (Vo)
Sample
  • Sample: Membrane sector (Vo) of yeast vacuolar ATPase
  • Protein or peptide: Vacuolar ATPase membrane sector
Keywordsyeast vacuolar ATPase / proton channel sector / reversible disassembly / membrane protein
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 18.0 Å
AuthorsCouoh-Cardel S / Milgrom E / Wilkens S
CitationJournal: J Biol Chem / Year: 2015
Title: Affinity Purification and Structural Features of the Yeast Vacuolar ATPase Vo Membrane Sector.
Authors: Sergio Couoh-Cardel / Elena Milgrom / Stephan Wilkens /
Abstract: The membrane sector (Vo) of the proton pumping vacuolar ATPase (V-ATPase, V1Vo-ATPase) from Saccharomyces cerevisiae was purified to homogeneity, and its structure was characterized by EM of single ...The membrane sector (Vo) of the proton pumping vacuolar ATPase (V-ATPase, V1Vo-ATPase) from Saccharomyces cerevisiae was purified to homogeneity, and its structure was characterized by EM of single molecules and two-dimensional crystals. Projection images of negatively stained Vo two-dimensional crystals showed a ring-like structure with a large asymmetric mass at the periphery of the ring. A cryo-EM reconstruction of Vo from single-particle images showed subunits a and d in close contact on the cytoplasmic side of the proton channel. A comparison of three-dimensional reconstructions of free Vo and Vo as part of holo V1Vo revealed that the cytoplasmic N-terminal domain of subunit a (aNT) must undergo a large conformational change upon enzyme disassembly or (re)assembly from Vo, V1, and subunit C. Isothermal titration calorimetry using recombinant subunit d and aNT revealed that the two proteins bind each other with a Kd of ~5 μm. Treatment of the purified Vo sector with 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] resulted in selective release of subunit d, allowing purification of a VoΔd complex. Passive proton translocation assays revealed that both Vo and VoΔd are impermeable to protons. We speculate that the structural change in subunit a upon release of V1 from Vo during reversible enzyme dissociation plays a role in blocking passive proton translocation across free Vo and that the interaction between aNT and d seen in free Vo functions to stabilize the Vo sector for efficient reassembly of V1Vo.
History
DepositionApr 7, 2015-
Header (metadata) releaseMay 6, 2015-
Map releaseOct 7, 2015-
UpdateNov 25, 2015-
Current statusNov 25, 2015Processing site: PDBe / 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 radius
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2975.tif

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Map

FileDownload / File: emd_2975.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of yeast V-ATPase membrane sector (Vo)
Voxel sizeX=Y=Z: 2.62 Å
Density
Contour LevelBy AUTHOR: 0.1 / Movie #1: 0.1
Minimum - Maximum-0.31523371 - 0.42559427
Average (Standard dev.)0.00741531 (±0.0690999)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-40-40-40
Dimensions808080
Spacing808080
CellA=B=C: 209.59999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.622.622.62
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z209.600209.600209.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-40-40-40
NC/NR/NS808080
D min/max/mean-0.3150.4260.007

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Supplemental data

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Sample components

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Entire : Membrane sector (Vo) of yeast vacuolar ATPase

EntireName: Membrane sector (Vo) of yeast vacuolar ATPase
Components
  • Sample: Membrane sector (Vo) of yeast vacuolar ATPase
  • Protein or peptide: Vacuolar ATPase membrane sector

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Supramolecule #1000: Membrane sector (Vo) of yeast vacuolar ATPase

SupramoleculeName: Membrane sector (Vo) of yeast vacuolar ATPase / type: sample / ID: 1000 / Details: Sample was monodisperse / Number unique components: 1
Molecular weightExperimental: 530 KDa / Theoretical: 320 KDa / Method: Small Angle X-ray Scattering

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Macromolecule #1: Vacuolar ATPase membrane sector

MacromoleculeName: Vacuolar ATPase membrane sector / type: protein_or_peptide / ID: 1 / Name.synonym: Vo
Details: V-ATPase membrane sector composed of subunits a, c, c', c'', d, e in the ratio 1:8:1:1:1:1
Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: vacuole
Molecular weightExperimental: 530 KDa / Theoretical: 320 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

Concentration2 mg/mL
BufferpH: 8
Details: 10 mM TRIS-HCl, 10 mM BME, 0.5 mM EGTA, 0.1 % dodecyl-beta-D-maltoside
GridDetails: 400 mesh holey carbon (C-flat 2/2) glow discharged in air
VitrificationCryogen name: ETHANE / Chamber temperature: 90 K / Instrument: HOMEMADE PLUNGER / Method: 5 sec blot in cold room

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Electron microscopy

MicroscopeJEOL 2100
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 57200 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 40000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 97 K / Max: 113 K / Average: 103 K
Alignment procedureLegacy - Astigmatism: Astigmatism was corrected at 200,000 times magnification over carbon film using live power spectrum
DateNov 10, 2010
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Digitization - Sampling interval: 15 µm / Average electron dose: 15 e/Å2 / Bits/pixel: 8

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Image processing

CTF correctionDetails: Each particle set (by micrograph)
Final two d classificationNumber classes: 217
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 18.0 Å / Resolution method: OTHER / Software - Name: IMAGIC, 5 / Number images used: 12035
DetailsParticle selection with EMAN1.9/Boxer; all subsequent image analysis with IMAGIC 5.
FSC plot (resolution estimation)

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