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- PDB-5tj5: Atomic model for the membrane-embedded motor of a eukaryotic V-ATPase -

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Basic information

Entry
Database: PDB / ID: 5tj5
TitleAtomic model for the membrane-embedded motor of a eukaryotic V-ATPase
Components(V-type proton ATPase subunit ...) x 7
KeywordsMOTOR PROTEIN / Rotary ATPase / Vacuolar-type ATPase / Electron Cryomicroscopy / Vo region / Membrane protein
Function / homology
Function and homology information


cell wall mannoprotein biosynthetic process / ATPase-coupled ion transmembrane transporter activity / cellular response to alkaline pH / polyphosphate metabolic process / P-type proton-exporting transporter activity / Insulin receptor recycling / Transferrin endocytosis and recycling / ROS and RNS production in phagocytes / Amino acids regulate mTORC1 / Golgi lumen acidification ...cell wall mannoprotein biosynthetic process / ATPase-coupled ion transmembrane transporter activity / cellular response to alkaline pH / polyphosphate metabolic process / P-type proton-exporting transporter activity / Insulin receptor recycling / Transferrin endocytosis and recycling / ROS and RNS production in phagocytes / Amino acids regulate mTORC1 / Golgi lumen acidification / vacuolar transport / vacuolar proton-transporting V-type ATPase, V0 domain / endosomal lumen acidification / protein targeting to vacuole / proton-transporting V-type ATPase complex / vacuolar proton-transporting V-type ATPase complex / vacuole organization / fungal-type vacuole / vacuolar acidification / cellular hyperosmotic response / fungal-type vacuole membrane / phosphatidylinositol-3,5-bisphosphate binding / proton transmembrane transporter activity / intracellular copper ion homeostasis / Neutrophil degranulation / proton-transporting ATPase activity, rotational mechanism / proton transmembrane transport / cell periphery / transmembrane transport / endocytosis / ATPase binding / protein-containing complex assembly / intracellular iron ion homeostasis / Golgi membrane / membrane
Similarity search - Function
lithium bound rotor ring of v- atpase / ATPase, V0 complex, subunit e1/e2 / ATP synthase subunit H / ATPase, V0 complex, subunit d / V-ATPase proteolipid subunit C, eukaryotic / ATPase, V0 complex, subunit 116kDa, eukaryotic / 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 ...lithium bound rotor ring of v- atpase / ATPase, V0 complex, subunit e1/e2 / ATP synthase subunit H / ATPase, V0 complex, subunit d / V-ATPase proteolipid subunit C, eukaryotic / ATPase, V0 complex, subunit 116kDa, eukaryotic / 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 / V-ATPase proteolipid subunit C-like domain / F/V-ATP synthase subunit C superfamily / ATP synthase subunit C / Four Helix Bundle (Hemerythrin (Met), subunit A) / Up-down Bundle / Mainly Alpha
Similarity search - Domain/homology
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 c' / V-type proton ATPase subunit e
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsMazhab-Jafari, M.T. / Rohou, A. / Schmidt, C. / Bueler, S.A. / Benlekbir, S. / Robinson, C.V. / Rubinstein, J.L.
Funding support Canada, United Kingdom, European Union, 4items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)MOP81294 Canada
Wellcome TrustWT008150 United Kingdom
Wellcome TrustWT099141 United Kingdom
European Research Council (ERC)ERC268851European Union
CitationJournal: Nature / Year: 2016
Title: Atomic model for the membrane-embedded V motor of a eukaryotic V-ATPase.
Authors: Mohammad T Mazhab-Jafari / Alexis Rohou / Carla Schmidt / Stephanie A Bueler / Samir Benlekbir / Carol V Robinson / John L Rubinstein /
Abstract: Vacuolar-type ATPases (V-ATPases) are ATP-powered proton pumps involved in processes such as endocytosis, lysosomal degradation, secondary transport, TOR signalling, and osteoclast and kidney ...Vacuolar-type ATPases (V-ATPases) are ATP-powered proton pumps involved in processes such as endocytosis, lysosomal degradation, secondary transport, TOR signalling, and osteoclast and kidney function. ATP hydrolysis in the soluble catalytic V region drives proton translocation through the membrane-embedded V region via rotation of a rotor subcomplex. Variability in the structure of the intact enzyme has prevented construction of an atomic model for the membrane-embedded motor of any rotary ATPase. We induced dissociation and auto-inhibition of the V and V regions of the V-ATPase by starving the yeast Saccharomyces cerevisiae, allowing us to obtain a ~3.9-Å resolution electron cryomicroscopy map of the V complex and build atomic models for the majority of its subunits. The analysis reveals the structures of subunits acc'c″de and a protein that we identify and propose to be a new subunit (subunit f). A large cavity between subunit a and the c-ring creates a cytoplasmic half-channel for protons. The c-ring has an asymmetric distribution of proton-carrying Glu residues, with the Glu residue of subunit c″ interacting with Arg735 of subunit a. The structure suggests sequential protonation and deprotonation of the c-ring, with ATP-hydrolysis-driven rotation causing protonation of a Glu residue at the cytoplasmic half-channel and subsequent deprotonation of a Glu residue at a luminal half-channel.
History
DepositionOct 3, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 26, 2016Provider: repository / Type: Initial release
Revision 1.1Nov 9, 2016Group: Database references
Revision 1.2Nov 16, 2016Group: Database references
Revision 1.3Sep 20, 2017Group: Author supporting evidence / Data collection / Category: em_software / pdbx_audit_support
Item: _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.4Mar 28, 2018Group: Data collection / Derived calculations / Category: pdbx_struct_assembly
Item: _pdbx_struct_assembly.details / _pdbx_struct_assembly.method_details
Revision 1.5Jan 15, 2020Group: Author supporting evidence / Category: pdbx_audit_support
Item: _pdbx_audit_support.country / _pdbx_audit_support.funding_organization
Revision 1.6Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: V-type proton ATPase subunit a
B: V-type proton ATPase subunit c''
D: V-type proton ATPase subunit c'
E: V-type proton ATPase subunit c
F: V-type proton ATPase subunit c
G: V-type proton ATPase subunit c
H: V-type proton ATPase subunit c
I: V-type proton ATPase subunit c
J: V-type proton ATPase subunit c
L: V-type proton ATPase subunit e
M: V-type proton ATPase subunit c
N: V-type proton ATPase subunit c
O: V-type proton ATPase subunit f
P: V-type proton ATPase subunit d


Theoretical massNumber of molelcules
Total (without water)273,56314
Polymers273,56314
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area33240 Å2
ΔGint-446 kcal/mol
Surface area104210 Å2

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Components

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V-type proton ATPase subunit ... , 7 types, 14 molecules ABDEFGHIJMNLOP

#1: Protein V-type proton ATPase subunit a / V-ATPase a 1 subunit / V-ATPase 95 kDa subunit / Vacuolar pH protein 1 / Vacuolar proton pump a ...V-ATPase a 1 subunit / V-ATPase 95 kDa subunit / Vacuolar pH protein 1 / Vacuolar proton pump a subunit / Vacuolar proton translocating ATPase subunit a 1


Mass: 70067.984 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P32563
#2: Protein V-type proton ATPase subunit c'' / V-ATPase subunit c'' / V-ATPase 22 kDa proteolipid subunit / Vacuolar proton pump c'' subunit


Mass: 22610.641 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P23968
#3: Protein V-type proton ATPase subunit c' / V-ATPase subunit c' / Proteolipid protein VMA11 / Trifluoperazine resistance protein 3 / V-ATPase ...V-ATPase subunit c' / Proteolipid protein VMA11 / Trifluoperazine resistance protein 3 / V-ATPase 16 kDa proteolipid subunit 2 / Vacuolar proton pump c' subunit


Mass: 15195.271 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P32842
#4: Protein
V-type proton ATPase subunit c / V-ATPase subunit c / Guanine nucleotide exchange factor 2 / V-ATPase 16 kDa proteolipid subunit 1 / ...V-ATPase subunit c / Guanine nucleotide exchange factor 2 / V-ATPase 16 kDa proteolipid subunit 1 / Vacuolar proton pump c subunit


Mass: 15218.087 Da / Num. of mol.: 8 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P25515
#5: Protein V-type proton ATPase subunit e / V-ATPase subunit e / Low dye-binding protein 10 / Vacuolar proton pump subunit e


Mass: 6476.928 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: Q3E7B6
#6: Protein V-type proton ATPase subunit f


Mass: 4613.678 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c
#7: Protein V-type proton ATPase subunit d / V-ATPase subunit d / V-ATPase 39 kDa subunit / V-ATPase subunit M39 / Vacuolar proton pump subunit d


Mass: 32854.238 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P32366

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Vo region of the V-ATPase from Saccharomyces cerevisiae
Type: COMPLEX / Entity ID: #1-#8 / Source: NATURAL
Molecular weightValue: 0.3 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.5
Details: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 micro M Bafilomycin
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The Vo region was solubilized in amphipol A8-35 (Anatrace)
Specimen supportGrid material: COPPER/RHODIUM / Grid mesh size: 400 divisions/in. / Grid type: Maxtaform
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277.3 K / Details: Modified for ethane/propane

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: 70 micro meter objective aperture, illuminated area of 1.58 micro meter diameter
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 37000 X / Calibrated magnification: 64350 X / Calibrated defocus min: 800 nm / Calibrated defocus max: 3400 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 21 sec. / Electron dose: 100 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4365
Image scansMovie frames/image: 70

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Processing

SoftwareName: PHENIX / Version: 1.10.1_2155: / Classification: refinement
EM software
IDNameVersionCategory
1RELION1.3particle selection
2SerialEMimage acquisition
4CTFFIND44.0.17CTF correction
7Coot0.8.2model fitting
9FREALIGN9.11initial Euler assignment
10FREALIGN9.11final Euler assignment
12FREALIGN9.113D reconstruction
19PHENIX1.10.1model refinement
Image processingDetails: Images were recorded in "super-resolution" mode and then resampled by Fourier cropping.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 657975
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 462842 / Algorithm: FOURIER SPACE
Details: Data beyond 6 A resolution were omitted from refinement.
Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Details: phenix.real_space_refine REFINEMENT TARGET: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS) MODEL TO MAP FIT. CC(ACROSS WHOLE MAP VOLUME): 0.6772 CC(ONLY ACROSS ATOMS IN THE MODEL): 0.6842
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0115174
ELECTRON MICROSCOPYf_angle_d1.24520815
ELECTRON MICROSCOPYf_dihedral_angle_d6.2410016
ELECTRON MICROSCOPYf_chiral_restr0.052636
ELECTRON MICROSCOPYf_plane_restr0.0062712

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