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- EMDB-5335: Sub-nanometer resolution structure of the intact T. thermophilus ... -

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Basic information

Entry
Database: EMDB / ID: EMD-5335
TitleSub-nanometer resolution structure of the intact T. thermophilus proton-driven ATP synthase reveals the arrangement of its trans-membrane helices
Map dataExperimental map of the V-type ATPase from Thermus thermophilus. Masks included are (a) density from subunit D not represented by the available crystal structure, (b) experimental map segment from subunit I, and (c) experimental map segment from L12-ring.
Sample
  • Sample: Thermus thermophilus V-type ATPase/ATP synthase solubilized with detergent
  • Protein or peptide: V-type ATPaseV-ATPase
KeywordsATP synthase / membrane protein complex / subnanometer / Thermus thermophilus / ATPase / V-ATPase
Function / homology
Function and homology information


proton-transporting V-type ATPase, V0 domain / proton-transporting two-sector ATPase complex, catalytic domain / proton-transporting ATP synthase complex / proton motive force-driven plasma membrane ATP synthesis / H+-transporting two-sector ATPase / proton-transporting ATPase activity, rotational mechanism / proton-transporting ATP synthase activity, rotational mechanism / ATP binding / metal ion binding
Similarity search - Function
ATPase, V0 complex, c subunit / Vacuolar (H+)-ATPase G subunit / ATPase, V1 complex, subunit F, bacterial/archaeal / 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 subunit E / V-type ATPase subunit E, C-terminal domain superfamily ...ATPase, V0 complex, c subunit / Vacuolar (H+)-ATPase G subunit / ATPase, V1 complex, subunit F, bacterial/archaeal / 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 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 / 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 / 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 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
V-type ATP synthase subunit D / V-type ATP synthase subunit E / V-type ATP synthase subunit C / V-type ATP synthase subunit F / V-type ATP synthase alpha chain / V-type ATP synthase beta chain / V-type ATP synthase, subunit (VAPC-THERM)
Similarity search - Component
Biological speciesThermus thermophilus HB8 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.7 Å
AuthorsLau WCY / Rubinstein JL
CitationJournal: Nature / Year: 2011
Title: Subnanometre-resolution structure of the intact Thermus thermophilus H+-driven ATP synthase.
Authors: Wilson C Y Lau / John L Rubinstein /
Abstract: Ion-translocating rotary ATPases serve either as ATP synthases, using energy from a transmembrane ion motive force to create the cell's supply of ATP, or as transmembrane ion pumps that are powered ...Ion-translocating rotary ATPases serve either as ATP synthases, using energy from a transmembrane ion motive force to create the cell's supply of ATP, or as transmembrane ion pumps that are powered by ATP hydrolysis. The members of this family of enzymes each contain two rotary motors: one that couples ion translocation to rotation and one that couples rotation to ATP synthesis or hydrolysis. During ATP synthesis, ion translocation through the membrane-bound region of the complex causes rotation of a central rotor that drives conformational changes and ATP synthesis in the catalytic region of the complex. There are no structural models available for the intact membrane region of any ion-translocating rotary ATPase. Here we present a 9.7 Å resolution map of the H(+)-driven ATP synthase from Thermus thermophilus obtained by electron cryomicroscopy of single particles in ice. The 600-kilodalton complex has an overall subunit composition of A(3)B(3)CDE(2)FG(2)IL(12). The membrane-bound motor consists of a ring of L subunits and the carboxy-terminal region of subunit I, which are equivalent to the c and a subunits of most other rotary ATPases, respectively. The map shows that the ring contains 12 L subunits and that the I subunit has eight transmembrane helices. The L(12) ring and I subunit have a surprisingly small contact area in the middle of the membrane, with helices from the I subunit making contacts with two different L subunits. The transmembrane helices of subunit I form bundles that could serve as half-channels across the membrane, with the first half-channel conducting protons from the periplasm to the L(12) ring and the second half-channel conducting protons from the L(12) ring to the cytoplasm. This structure therefore suggests the mechanism by which a transmembrane proton motive force is converted to rotation in rotary ATPases.
History
DepositionAug 30, 2011-
Header (metadata) releaseDec 8, 2011-
Map releaseDec 8, 2011-
UpdateMay 31, 2012-
Current statusMay 31, 2012Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.548
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.548
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j0j
  • Surface level: 0.548
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3j0j
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5335_1.png

Downloads & links

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Map

FileDownload / File: emd_5335.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationExperimental map of the V-type ATPase from Thermus thermophilus. Masks included are (a) density from subunit D not represented by the available crystal structure, (b) experimental map segment from subunit I, and (c) experimental map segment from L12-ring.
Voxel sizeX=Y=Z: 1.4 Å
Density
Contour LevelBy AUTHOR: 0.548 / Movie #1: 0.548
Minimum - Maximum-0.4174926 - 1.57104111
Average (Standard dev.)0.01590016 (±0.13230292)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-128-128-128
Dimensions256256256
Spacing256256256
CellA=B=C: 358.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.41.41.4
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z358.400358.400358.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-62-62-62
NX/NY/NZ125125125
MAP C/R/S123
start NC/NR/NS-128-128-128
NC/NR/NS256256256
D min/max/mean-0.4171.5710.016

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Supplemental data

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Segmentation: Experimental map segment from subunit I

AnnotationExperimental map segment from subunit I
Fileemd_5335_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Segmentation: Experimental map segment from L12-ring

AnnotationExperimental map segment from L12-ring
Fileemd_5335_msk_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Segmentation: Density from subunit D not represented by the...

AnnotationDensity from subunit D not represented by the available crystal structure
Fileemd_5335_msk_3.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Thermus thermophilus V-type ATPase/ATP synthase solubilized with ...

EntireName: Thermus thermophilus V-type ATPase/ATP synthase solubilized with detergent
Components
  • Sample: Thermus thermophilus V-type ATPase/ATP synthase solubilized with detergent
  • Protein or peptide: V-type ATPaseV-ATPase

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Supramolecule #1000: Thermus thermophilus V-type ATPase/ATP synthase solubilized with ...

SupramoleculeName: Thermus thermophilus V-type ATPase/ATP synthase solubilized with detergent
type: sample / ID: 1000 / Details: Solubilized with the detergent dodecyl maltoside / Oligomeric state: Monomeric / Number unique components: 1
Molecular weightExperimental: 600 KDa / Theoretical: 600 KDa

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Macromolecule #1: V-type ATPase

MacromoleculeName: V-type ATPase / type: protein_or_peptide / ID: 1 / Name.synonym: V-type ATPase / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Thermus thermophilus HB8 (bacteria) / synonym: Thermus thermophilus / Cell: Thermus thermophilus HB8 / Organelle: plasma membrane / Location in cell: plasma membrane
Molecular weightTheoretical: 600 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

Concentration2.5 mg/mL
BufferpH: 8
Details: 50mM Tris-HCl, 5mM MgCl2, 150 mM NaCl, 0.02% (w/v) dodecyl maltoside
GridDetails: 200 mesh quantifoil
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 95 K / Instrument: FEI VITROBOT MARK III / Details: Vitrification instrument: Vitrobot Mark 3 / Method: Blot for 20 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Gatan 626 / Specimen holder model: GATAN LIQUID NITROGEN
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism corrected around 100,000x magnification
DateNov 3, 2001
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 700 / Average electron dose: 18 e/Å2 / Bits/pixel: 8
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: 9.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Frealign, Refine_Fspace, Build_FSpace
Details: Map constructed in Fourier space by Build_Fspace, sharpened with an inverse temperature factor of 750 Angstroms squared
Number images used: 46105

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Atomic model buiding 1

Initial modelPDB ID:

3a5c


Chain - #0 - Chain ID: I / Chain - #1 - Chain ID: J / Chain - #2 - Chain ID: K / Chain - #3 - Chain ID: L / Chain - #4 - Chain ID: M / Chain - #5 - Chain ID: N / Chain - #6 - Chain ID: O / Chain - #7 - Chain ID: P
SoftwareName: UCSF Chimera
DetailsPDBEntryID_givenInChain. Protocol: Rigid Body. V1 subcomplex crystal structure
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient
Output model

PDB-3j0j:
Fitted atomic models of Thermus thermophilus V-ATPase subunits into cryo-EM map

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Atomic model buiding 2

Initial modelPDB ID:

1r5z


Chain - Chain ID: A
SoftwareName: Imodfit
DetailsPDBEntryID_givenInChain. Protocol: Flexible. C subunit crystal structure
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-3j0j:
Fitted atomic models of Thermus thermophilus V-ATPase subunits into cryo-EM map

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Atomic model buiding 3

Initial modelPDB ID:

3k5b


Chain - #0 - Chain ID: G / Chain - #1 - Chain ID: E
SoftwareName: UCSF Chimera
DetailsPDBEntryID_givenInChain. Protocol: Rigid Body. Two copies of this peripheral stalk (EG subcomplex) structure fitted into the cryo-EM map because there are two copies in the complex.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient
Output model

PDB-3j0j:
Fitted atomic models of Thermus thermophilus V-ATPase subunits into cryo-EM map

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