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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-6285 | |||||||||
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Title | Yeast V-ATPase state 2 | |||||||||
![]() | Yeast V-ATPase state 2 | |||||||||
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![]() | V-ATPase / V-type ATPase / vacuolar-type ATPase / yeast / Saccharomyces cerevisiae / hydrolase | |||||||||
Function / homology | ![]() vacuole-mitochondrion membrane contact site / protein localization to vacuolar membrane / cellular response to alkaline pH / Insulin receptor recycling / Transferrin endocytosis and recycling / polyphosphate metabolic process / ROS and RNS production in phagocytes / Amino acids regulate mTORC1 / Golgi lumen acidification / proteasome storage granule assembly ...vacuole-mitochondrion membrane contact site / protein localization to vacuolar membrane / cellular response to alkaline pH / Insulin receptor recycling / Transferrin endocytosis and recycling / polyphosphate metabolic process / ROS and RNS production in phagocytes / Amino acids regulate mTORC1 / Golgi lumen acidification / proteasome storage granule assembly / pexophagy / endosomal lumen acidification / vacuolar proton-transporting V-type ATPase, V0 domain / vacuolar proton-transporting V-type ATPase, V1 domain / vacuolar transport / protein targeting to vacuole / vacuole organization / proton-transporting V-type ATPase complex / protein metabolic process / fungal-type vacuole / intein-mediated protein splicing / intron homing / cellular hyperosmotic response / vacuolar proton-transporting V-type ATPase complex / phosphatidylinositol-3,5-bisphosphate binding / vacuolar acidification / fungal-type vacuole membrane / 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 / membrane raft / Golgi membrane / mRNA binding / ATP hydrolysis activity / DNA binding / ATP binding / membrane / cytoplasm Similarity search - Function | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 7.6 Å | |||||||||
![]() | Zhao J / Benlekbir S / Rubinstein JL | |||||||||
![]() | ![]() 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 |
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Structure visualization
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 57.3 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 13.7 KB 13.7 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 11 KB | Display | ![]() |
Images | ![]() | 112.2 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 3j9uMC ![]() 6284C ![]() 6286C ![]() 3j9tC ![]() 3j9vC C: citing same article ( M: atomic model generated by this map |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | Yeast V-ATPase state 2 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.45 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Vacuolar-type ATPase
Entire | Name: Vacuolar-type ATPase |
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Components |
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-Supramolecule #1000: Vacuolar-type ATPase
Supramolecule | Name: 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 |
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Molecular weight | Experimental: 900 KDa / Theoretical: 900 KDa / Method: SDS-PAGE, size exclusion chromatography |
-Macromolecule #1: Vacuolar-type ATPase
Macromolecule | Name: 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 |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Experimental: 900 KDa / Theoretical: 900 KDa |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 10 mg/mL |
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Buffer | pH: 7.4 Details: 50 mM Tris-HCl, 150 mM NaCl, 0.02% w/v dodecylmaltoside |
Grid | Details: Homemade holey carbon on 400 square mesh Cu/Rh grid, glow-discharged 2 mins |
Vitrification | Cryogen 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
Microscope | FEI TECNAI F20 |
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Alignment procedure | Legacy - Astigmatism: Manually corrected by inspecting FFT. |
Details | K2 Summit in counting mode, 2 frames/s for 15 s |
Date | Oct 19, 2013 |
Image recording | Category: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 3685 / Average electron dose: 30 e/Å2 / Bits/pixel: 32 |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | Calibrated magnification: 34483 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 7.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 34483 |
Sample stage | Specimen holder model: GATAN LIQUID NITROGEN |
Experimental equipment | ![]() Model: Tecnai F20 / Image courtesy: FEI Company |
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Image processing
-Atomic model buiding 1
Initial model | PDB ID: Chain - Chain ID: A |
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Software | Name: Chimera, MDFF |
Details | Rigid body fitting performed in Chimera first, followed by flexible fitting performed using Molecular Dynamics Flexible Fitting (MDFF). |
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |
Output model | ![]() PDB-3j9u: |
-Atomic model buiding 2
Initial model | PDB ID: Chain - Chain ID: A |
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Software | Name: Chimera, MDFF |
Details | Rigid body fitting performed in Chimera first, followed by flexible fitting performed using Molecular Dynamics Flexible Fitting (MDFF). |
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |
Output model | ![]() PDB-3j9u: |
-Atomic model buiding 3
Initial model | PDB ID: Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B |
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Software | Name: Chimera, MDFF |
Details | Rigid body fitting performed in Chimera first, followed by flexible fitting performed using Molecular Dynamics Flexible Fitting (MDFF). |
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |
Output model | ![]() PDB-3j9u: |
-Atomic model buiding 4
Initial model | PDB ID: Chain - #0 - Chain ID: E / Chain - #1 - Chain ID: G |
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Software | Name: Chimera, MDFF |
Details | Rigid body fitting performed in Chimera first, followed by flexible fitting performed using Molecular Dynamics Flexible Fitting (MDFF). |
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |
Output model | ![]() PDB-3j9u: |