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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-9661 | |||||||||
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Title | 17A ZPC cryoEM map of Eh V-ATPase | |||||||||
![]() | 17A ZPC cryoEM map of EhV-ATPase | |||||||||
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Method | single particle reconstruction / cryo EM / Resolution: 17.0 Å | |||||||||
![]() | Tsunoda J / Song C / Imai FL / Murata T / Ueno H / Iino R / Takagi J / Murata K | |||||||||
![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 28.2 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 12.1 KB 12.1 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 6.9 KB | Display | ![]() |
Images | ![]() | 44.7 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
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Links
EMDB pages | ![]() ![]() |
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Map
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Annotation | 17A ZPC cryoEM map of EhV-ATPase | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.992 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 : Enterococcus hirae V-ATPse
Entire | Name: Enterococcus hirae V-ATPse |
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Components |
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-Supramolecule #1: Enterococcus hirae V-ATPse
Supramolecule | Name: Enterococcus hirae V-ATPse / type: complex / ID: 1 / Parent: 0 |
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Recombinant expression | Organism: ![]() ![]() |
Molecular weight | Experimental: 800 kDa/nm |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.5 |
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Sugar embedding | Material: ice |
Grid | Model: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: MOLYBDENUM / Mesh: 400 |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
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Electron microscopy
Microscope | JEOL 2200FS |
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Temperature | Min: 76.0 K / Max: 76.0 K |
Specialist optics | Phase plate: ZERNIKE PHASE PLATE / Energy filter - Name: In-column Omega Filter / Energy filter - Slit width: 20 eV |
Image recording | Film 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: 3 / Number real images: 665 / Average exposure time: 3.0 sec. / Average electron dose: 20.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | C2 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 FIELD / Cs: 4.2 mm / Nominal defocus max: 0.5 µm / Nominal defocus min: 0.25 µm / Nominal magnification: 30000 |
Sample stage | Specimen holder model: GATAN 914 HIGH TILT LIQUID NITROGEN CRYO TRANSFER TOMOGRAPHY HOLDER Cooling holder cryogen: NITROGEN |