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- EMDB-6284: Yeast V-ATPase state 1 -

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Basic information

Entry
Database: EMDB / ID: EMD-6284
TitleYeast V-ATPase state 1
Map dataYeast V-ATPase state 1
Sample
  • Sample: Vacuolar-type ATPase
  • Protein or peptide: Vacuolar-type ATPase
KeywordsV-ATPase / V-type ATPase / vacuolar-type ATPase / yeast / Saccharomyces cerevisiae / hydrolase
Function / homology
Function and homology information


vacuole-mitochondrion membrane contact site / cellular response to alkaline pH / polyphosphate metabolic process / Insulin receptor recycling / Transferrin endocytosis and recycling / ROS and RNS production in phagocytes / Amino acids regulate mTORC1 / Golgi lumen acidification / proteasome storage granule assembly / vacuolar proton-transporting V-type ATPase, V1 domain ...vacuole-mitochondrion membrane contact site / cellular response to alkaline pH / polyphosphate metabolic process / Insulin receptor recycling / Transferrin endocytosis and recycling / ROS and RNS production in phagocytes / Amino acids regulate mTORC1 / Golgi lumen acidification / proteasome storage granule assembly / vacuolar proton-transporting V-type ATPase, V1 domain / vacuolar transport / vacuolar proton-transporting V-type ATPase, V0 domain / endosomal lumen acidification / pexophagy / protein targeting to vacuole / proton-transporting V-type ATPase complex / vacuolar proton-transporting V-type ATPase complex / vacuole organization / fungal-type vacuole / vacuolar acidification / intron homing / cellular hyperosmotic response / protein metabolic process / intein-mediated protein splicing / fungal-type vacuole membrane / phosphatidylinositol-3,5-bisphosphate binding / proton transmembrane transporter activity / intracellular copper ion homeostasis / ATP metabolic process / H+-transporting two-sector ATPase / Neutrophil degranulation / proton-transporting ATPase activity, rotational mechanism / proton transmembrane transport / proton-transporting ATP synthase activity, rotational mechanism / transmembrane transport / intracellular calcium ion homeostasis / cytoplasmic stress granule / endocytosis / ATPase binding / protein-containing complex assembly / endonuclease activity / intracellular iron ion homeostasis / Hydrolases; Acting on ester bonds / Golgi membrane / mRNA binding / DNA binding / ATP binding / membrane / cytoplasm
Similarity search - Function
Homing endonuclease PI-Sce / Homing endonuclease / Hom-end-associated Hint / Hom_end-associated Hint / ATPase, V1 complex, subunit H / ATPase, V1 complex, subunit H, C-terminal / ATPase, V1 complex, subunit H, C-terminal domain superfamily / V-ATPase subunit H / V-ATPase subunit H / ATPase, V1 complex, subunit C ...Homing endonuclease PI-Sce / Homing endonuclease / Hom-end-associated Hint / Hom_end-associated Hint / ATPase, V1 complex, subunit H / ATPase, V1 complex, subunit H, C-terminal / ATPase, V1 complex, subunit H, C-terminal domain superfamily / V-ATPase subunit H / V-ATPase subunit H / ATPase, V1 complex, subunit C / Vacuolar ATP synthase subunit C superfamily / V-ATPase subunit C / Vacuolar (H+)-ATPase G subunit / Vacuolar (H+)-ATPase G subunit / ATPase, V1 complex, subunit B / ATPase, V1 complex, subunit F, eukaryotic / ATPase, V0 complex, subunit d / V-ATPase proteolipid subunit C, eukaryotic / ATPase, V0 complex, subunit 116kDa, eukaryotic / Intein / V-ATPase proteolipid subunit / ATPase, V0 complex, c/d subunit / V-type ATPase subunit C/d / V-type ATP synthase subunit c/d subunit superfamily / V-type ATP synthase c/d subunit, domain 3 superfamily / ATP synthase (C/AC39) subunit / V-type ATPase, V0 complex, 116kDa subunit family / V-type ATPase 116kDa subunit family / Intein DOD homing endonuclease / Intein DOD-type homing endonuclease domain profile. / Intein C-terminal splicing region / Intein C-terminal splicing motif profile. / V-type ATPase subunit E / V-type ATPase subunit E, C-terminal domain superfamily / ATP synthase (E/31 kDa) subunit / ATPase, V1 complex, subunit D / ATPase, V1 complex, subunit F / ATPase, V1 complex, subunit F superfamily / ATP synthase subunit D / ATP synthase (F/14-kDa) subunit / Hint domain C-terminal / Hint (Hedgehog/Intein) domain C-terminal region / V-type ATP synthase regulatory subunit B/beta / V-type ATP synthase catalytic alpha chain / ATPsynthase alpha/beta subunit, N-terminal extension / ATPsynthase alpha/beta subunit N-term extension / Intein N-terminal splicing region / Intein N-terminal splicing motif profile. / Hint domain N-terminal / Hint (Hedgehog/Intein) domain N-terminal region / Hint domain superfamily / Homing endonuclease / V-ATPase proteolipid subunit C-like domain / F/V-ATP synthase subunit C superfamily / ATP synthase subunit C / ATPase, F1/V1 complex, beta/alpha subunit, C-terminal / ATP synthase subunit alpha, N-terminal domain-like superfamily / ATPase, F1/V1/A1 complex, alpha/beta subunit, N-terminal domain superfamily / ATPase, F1/V1/A1 complex, alpha/beta subunit, N-terminal domain / ATP synthase alpha/beta family, beta-barrel domain / ATPase, alpha/beta subunit, nucleotide-binding domain, active site / ATP synthase alpha and beta subunits signature. / ATPase, F1/V1/A1 complex, alpha/beta subunit, nucleotide-binding domain / ATP synthase alpha/beta family, nucleotide-binding domain / Armadillo-like helical / Armadillo-type fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
V-type proton ATPase subunit B / V-type proton ATPase catalytic subunit A / V-type proton ATPase subunit E / V-type proton ATPase subunit c / V-type proton ATPase subunit C / V-type proton ATPase subunit d / V-type proton ATPase subunit a, vacuolar isoform / V-type proton ATPase subunit D / V-type proton ATPase subunit F / V-type proton ATPase subunit H / V-type proton ATPase subunit G
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.9 Å
AuthorsZhao J / Benlekbir S / Rubinstein JL
CitationJournal: Nature / Year: 2015
Title: Electron cryomicroscopy observation of rotational states in a eukaryotic V-ATPase.
Authors: Jianhua Zhao / Samir Benlekbir / John L Rubinstein /
Abstract: Eukaryotic vacuolar H(+)-ATPases (V-ATPases) are rotary enzymes that use energy from hydrolysis of ATP to ADP to pump protons across membranes and control the pH of many intracellular compartments. ...Eukaryotic vacuolar H(+)-ATPases (V-ATPases) are rotary enzymes that use energy from hydrolysis of ATP to ADP to pump protons across membranes and control the pH of many intracellular compartments. ATP hydrolysis in the soluble catalytic region of the enzyme is coupled to proton translocation through the membrane-bound region by rotation of a central rotor subcomplex, with peripheral stalks preventing the entire membrane-bound region from turning with the rotor. The eukaryotic V-ATPase is the most complex rotary ATPase: it has three peripheral stalks, a hetero-oligomeric proton-conducting proteolipid ring, several subunits not found in other rotary ATPases, and is regulated by reversible dissociation of its catalytic and proton-conducting regions. Studies of ATP synthases, V-ATPases, and bacterial/archaeal V/A-ATPases have suggested that flexibility is necessary for the catalytic mechanism of rotary ATPases, but the structures of different rotational states have never been observed experimentally. Here we use electron cryomicroscopy to obtain structures for three rotational states of the V-ATPase from the yeast Saccharomyces cerevisiae. The resulting series of structures shows ten proteolipid subunits in the c-ring, setting the ATP:H(+) ratio for proton pumping by the V-ATPase at 3:10, and reveals long and highly tilted transmembrane α-helices in the a-subunit that interact with the c-ring. The three different maps reveal the conformational changes that occur to couple rotation in the symmetry-mismatched soluble catalytic region to the membrane-bound proton-translocating region. Almost all of the subunits of the enzyme undergo conformational changes during the transitions between these three rotational states. The structures of these states provide direct evidence that deformation during rotation enables the smooth transmission of power through rotary ATPases.
History
DepositionFeb 22, 2015-
Header (metadata) releaseMar 18, 2015-
Map releaseMay 13, 2015-
UpdateAug 26, 2015-
Current statusAug 26, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j9t
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3j9t
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6284.png

Downloads & links

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Map

FileDownload / File: emd_6284.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationYeast V-ATPase state 1
Voxel sizeX=Y=Z: 1.45 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.04820368 - 0.15646601
Average (Standard dev.)0.00046598 (±0.00966013)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 371.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.451.451.45
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z371.200371.200371.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0480.1560.000

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Supplemental data

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Sample components

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Entire : Vacuolar-type ATPase

EntireName: Vacuolar-type ATPase
Components
  • Sample: Vacuolar-type ATPase
  • Protein or peptide: Vacuolar-type ATPase

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Supramolecule #1000: Vacuolar-type ATPase

SupramoleculeName: Vacuolar-type ATPase / type: sample / ID: 1000 / Details: Detergent solubilized protein complex
Oligomeric state: A3B3CDE3FG3HadcXc'Yc''Z where X, Y, and Z indicate unknown stoichiometry and X+Y+Z=10
Number unique components: 1
Molecular weightExperimental: 900 KDa / Theoretical: 900 KDa / Method: SDS-PAGE, size exclusion chromatography

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Macromolecule #1: Vacuolar-type ATPase

MacromoleculeName: Vacuolar-type ATPase / type: protein_or_peptide / ID: 1 / Name.synonym: V-ATPase, V-type ATPase / Details: Detergent-solubilized protein complex / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: SABY31 / synonym: Yeast
Molecular weightExperimental: 900 KDa / Theoretical: 900 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

Concentration10 mg/mL
BufferpH: 7.4
Details: 50 mM Tris-HCl, 150 mM NaCl, 0.02% w/v dodecylmaltoside
GridDetails: Homemade holey carbon on 400 square mesh Cu/Rh grid, glow-discharged 2 mins
VitrificationCryogen name: ETHANE-PROPANE MIXTURE / Chamber humidity: 100 % / Chamber temperature: 77 K / Instrument: FEI VITROBOT MARK III / Method: Blot for 23 seconds before freezing.

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 34483 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 7.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 34483
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Alignment procedureLegacy - Astigmatism: Manually corrected by inspecting FFT.
DetailsK2 Summit in counting mode, 2 frames/s for 15 s
DateOct 19, 2013
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 3685 / Average electron dose: 30 e/Å2 / Bits/pixel: 32
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 6.9 Å / Resolution method: OTHER / Software - Name: Relion / Number images used: 50503
DetailsParticles automatically selected using TMaCS and processed in Relion.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

1u7l


Chain - Chain ID: A
SoftwareName: Chimera, MDFF
DetailsRigid body fitting performed in Chimera first, followed by flexible fitting performed using Molecular Dynamics Flexible Fitting (MDFF).
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-3j9t:
Yeast V-ATPase state 1

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Atomic model buiding 2

Initial modelPDB ID:

1ho8


Chain - Chain ID: A
SoftwareName: Chimera, MDFF
DetailsRigid body fitting performed in Chimera first, followed by flexible fitting performed using Molecular Dynamics Flexible Fitting (MDFF).
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-3j9t:
Yeast V-ATPase state 1

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Atomic model buiding 3

Initial modelPDB ID:

4rnd


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B
SoftwareName: Chimera, MDFF
DetailsRigid body fitting performed in Chimera first, followed by flexible fitting performed using Molecular Dynamics Flexible Fitting (MDFF).
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-3j9t:
Yeast V-ATPase state 1

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Atomic model buiding 4

Initial modelPDB ID:

4dl0


Chain - #0 - Chain ID: E / Chain - #1 - Chain ID: G
SoftwareName: Chimera, MDFF
DetailsRigid body fitting performed in Chimera first, followed by flexible fitting performed using Molecular Dynamics Flexible Fitting (MDFF).
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-3j9t:
Yeast V-ATPase state 1

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