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Open data
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Basic information
Entry | Database: PDB / ID: 3j9t | ||||||
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Title | Yeast V-ATPase state 1 | ||||||
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![]() | HYDROLASE / V-ATPase / V-type ATPase / vacuolar-type ATPase / proton pump | ||||||
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 | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.9 Å | ||||||
![]() | Zhao, J. / Benlekbir, S. / Rubinstein, J.L. | ||||||
![]() | ![]() 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. | ||||||
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Structure visualization
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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PDBx/mmCIF format | ![]() | 1.2 MB | Display | ![]() |
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PDB format | ![]() | 976.7 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 6284MC ![]() 6285C ![]() 6286C ![]() 3j9uC ![]() 3j9vC M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Assembly
Deposited unit | ![]()
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Components
-V-type proton ATPase subunit ... , 10 types, 25 molecules MNBDFQLHJKGIPbOYRUVTWSXZa
#1: Protein | Mass: 29235.023 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() | ||||||||||||||
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#2: Protein | Mass: 13479.170 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() | ||||||||||||||
#4: Protein | Mass: 57815.023 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() #5: Protein | | Mass: 39822.484 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() #6: Protein | Mass: 12738.706 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() #7: Protein | Mass: 26508.393 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() #8: Protein | | Mass: 54482.609 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() #9: Protein | | Mass: 95625.484 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() #10: Protein | | Mass: 44241.352 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() #11: Protein | Mass: 16357.501 Da / Num. of mol.: 10 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
-Protein , 1 types, 3 molecules ACE
#3: Protein | Mass: 67665.305 Da / Num. of mol.: 3 / Fragment: SEE REMARK 999 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() References: UniProt: P17255, H+-transporting two-sector ATPase, Hydrolases; Acting on ester bonds |
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-Details
Sequence details | CHAINS A, C, AND E CONTAIN RESIDUES 2-283 and 738-1071 OF UNP P17255 WITH RESIDUES 284-737 REMOVED. |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: Vacuolar-type ATPase / Type: COMPLEX Details: A3B3CDE3FG3HadcXc'Yc''Z where X, Y, and Z indicate unknown stoichiometry and X+Y+Z=10 |
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Molecular weight | Value: 0.9 MDa / Experimental value: YES |
Buffer solution | Name: 50 mM Tris-HCl, 150 mM NaCl, 0.02% w/v dodecylmaltoside pH: 7.4 Details: 50 mM Tris-HCl, 150 mM NaCl, 0.02% w/v dodecylmaltoside |
Specimen | Conc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Details: Homemade holey carbon on 400 square mesh Cu/Rh grid, glow-discharged 2 mins |
Vitrification | Instrument: FEI VITROBOT MARK III / Cryogen name: OTHER / Temp: 77 K / Humidity: 100 % Details: Blot for 23 seconds before freezing in ethane/propane mixture (FEI VITROBOT MARK III). Method: Blot for 23 seconds before freezing |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Tecnai F20 / Image courtesy: FEI Company |
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Microscopy | Model: FEI TECNAI F20 / Date: Oct 19, 2013 / Details: K2 Summit in counting mode, 2 frames/s for 15 s |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 34483 X / Calibrated magnification: 34483 X / Nominal defocus max: 7000 nm / Nominal defocus min: 1500 nm / Cs: 2 mm / Astigmatism: Manually corrected by inspecting FFT |
Specimen holder | Specimen holder model: GATAN LIQUID NITROGEN / Temperature: 77 K |
Image recording | Electron dose: 30 e/Å2 / Film or detector model: GATAN K2 (4k x 4k) / Details: K2 Summit in counting mode |
Image scans | Num. digital images: 3685 |
Radiation | Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray |
Radiation wavelength | Relative weight: 1 |
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Processing
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CTF correction | Details: Each particle | |||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | |||||||||||||||||||||||||||||||||||
3D reconstruction | Method: Maximum likelihood / Resolution: 6.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 50503 / Nominal pixel size: 1.45 Å / Actual pixel size: 1.45 Å Details: (Single particle details: Particles automatically selected using TMaCS and processed in Relion.) (Single particle--Applied symmetry: C1) Symmetry type: POINT | |||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL Details: REFINEMENT PROTOCOL--flexible DETAILS--Rigid body fitting performed in Chimera first, followed by flexible fitting performed using Molecular Dynamics Flexible Fitting (MDFF). | |||||||||||||||||||||||||||||||||||
Atomic model building | 3D fitting-ID: 1 / Source name: PDB / Type: experimental model
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Refinement step | Cycle: LAST
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