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- EMDB-9662: 19A ZPC cryo-EM map of Eh V-ATPase-Fab -

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Basic information

Entry
Database: EMDB / ID: EMD-9662
Title19A ZPC cryo-EM map of Eh V-ATPase-Fab
Map data19A ZPC cryo-EM map of Eh V-ATPase-Fab
Sample
  • Complex: Enterococcus hirae V-ATPse
Methodsingle particle reconstruction / cryo EM / Resolution: 19.0 Å
AuthorsTsunoda J / Song C / Imai FL / Murata T / Ueno H / Iino R / Takagi J / Murata K
CitationJournal: Sci Rep / Year: 2018
Title: Off-axis rotor in Enterococcus hirae V-ATPase visualized by Zernike phase plate single-particle cryo-electron microscopy.
Authors: Jun Tsunoda / Chihong Song / Fabiana Lica Imai / Junichi Takagi / Hiroshi Ueno / Takeshi Murata / Ryota Iino / Kazuyoshi Murata /
Abstract: EhV-ATPase is an ATP-driven Na pump in the eubacteria Enterococcus hirae (Eh). Here, we present the first entire structure of detergent-solubilized EhV-ATPase by single-particle cryo-electron ...EhV-ATPase is an ATP-driven Na pump in the eubacteria Enterococcus hirae (Eh). Here, we present the first entire structure of detergent-solubilized EhV-ATPase by single-particle cryo-electron microscopy (cryo-EM) using Zernike phase plate. The cryo-EM map dominantly showed one of three catalytic conformations in this rotary enzyme. To further stabilize the originally heterogeneous structure caused by the ATP hydrolysis states of the V-ATPases, a peptide epitope tag system was adopted, in which the inserted peptide epitope sequence interfered with rotation of the central rotor by binding the Fab. As a result, the map unexpectedly showed another catalytic conformation of EhV-ATPase. Interestingly, these two conformations identified with and without Fab conversely coincided with those of the minor state 2 and the major state 1 of Thermus thermophilus V/A-ATPase, respectively. The most prominent feature in EhV-ATPase was the off-axis rotor, where the cytoplasmic V domain was connected to the transmembrane V domain through the off-axis central rotor. Furthermore, compared to the structure of ATP synthases, the larger size of the interface between the transmembrane a-subunit and c-ring of EhV-ATPase would be more advantageous for active ion pumping.
History
DepositionSep 23, 2018-
Header (metadata) releaseNov 7, 2018-
Map releaseNov 7, 2018-
UpdateAug 5, 2020-
Current statusAug 5, 2020Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0262
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0262
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9662.png

Downloads & links

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Map

FileDownload / File: emd_9662.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation19A ZPC cryo-EM map of Eh V-ATPase-Fab
Voxel sizeX=Y=Z: 1.992 Å
Density
Contour LevelBy AUTHOR: 0.0262 / Movie #1: 0.0262
Minimum - Maximum-0.04633683 - 0.08095652
Average (Standard dev.)0.0013634621 (±0.00937568)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 398.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.9921.9921.992
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z398.400398.400398.400
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ352352352
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0460.0810.001

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Supplemental data

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Sample components

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Entire : Enterococcus hirae V-ATPse

EntireName: Enterococcus hirae V-ATPse
Components
  • Complex: Enterococcus hirae V-ATPse

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Supramolecule #1: Enterococcus hirae V-ATPse

SupramoleculeName: Enterococcus hirae V-ATPse / type: complex / ID: 1 / Parent: 0
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightExperimental: 800 kDa/nm

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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

BufferpH: 7.5
Sugar embeddingMaterial: ice
GridModel: Quantifoil R1.2/1.3 / Material: MOLYBDENUM / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeJEOL 2200FS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 40.0 µm / Calibrated defocus max: 0.5 µm / Calibrated defocus min: 0.25 µm / Calibrated magnification: 32129 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 4.2 mm / Nominal defocus max: 0.5 µm / Nominal defocus min: 0.25 µm / Nominal magnification: 30000
Specialist opticsPhase plate: ZERNIKE PHASE PLATE / Energy filter - Name: In-column Omega Filter / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: GATAN 914 HIGH TILT LIQUID NITROGEN CRYO TRANSFER TOMOGRAPHY HOLDER
Cooling holder cryogen: NITROGEN
TemperatureMin: 76.0 K / Max: 76.0 K
Image recordingFilm or detector model: DIRECT ELECTRON DE-20 (5k x 3k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 5120 pixel / Digitization - Dimensions - Height: 3840 pixel / Digitization - Sampling interval: 6.4 µm / Digitization - Frames/image: 3-75 / Number grids imaged: 5 / Number real images: 1302 / Average exposure time: 3.0 sec. / Average electron dose: 20.0 e/Å2

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Image processing

Particle selectionNumber selected: 114509
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final 3D classificationNumber classes: 12 / Avg.num./class: 5000 / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 19.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 35413
FSC plot (resolution estimation)

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