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- EMDB-31312: A3B3 ring of V1-ATPase on EG-grid -

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Basic information

Entry
Database: EMDB / ID: EMD-31312
TitleA3B3 ring of V1-ATPase on EG-grid
Map data
Sample
  • Complex: The chimeric complex of A3B3 ring of V-ATPase from Thermus thermophilus and DF subunit of V-ATPase from human.
    • Complex: The chimeric complex of A3B3 ring from Thermus thermophilus
      • Protein or peptide: A subunit of V1-ATPase from Thermus thermophilus.
    • Complex: DF subunit from human
      • Protein or peptide: B subunit of V1-ATPase from Thermus thermophilus.
Biological speciesThermus thermophilus (unknown) / Homo sapiens (human) / Thermus thermophilus HB8 (unknown)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.03 Å
AuthorsFujita J / Makino F / Asahara H / Moriguchi M / Kumano S / Anzai I / Kishikawa J / Matsuura Y / Kato T / Namba K / Inoue T
Funding support Japan, 7 items
OrganizationGrant numberCountry
Japan Science and TechnologyJPMJOP1861 Japan
New Energy and Industrial Technology Development Organization (NEDO)16102003-0 Japan
New Energy and Industrial Technology Development Organization (NEDO)17101509-0 Japan
Japan Society for the Promotion of Science (JSPS)JP25000013 Japan
Japan Society for the Promotion of Science (JSPS)JP20K22630 Japan
Japan Agency for Medical Research and Development (AMED)JP19am0101117 Japan
Japan Agency for Medical Research and Development (AMED)JP17pc0101020 Japan
CitationJournal: Sci Rep / Year: 2023
Title: Epoxidized graphene grid for highly efficient high-resolution cryoEM structural analysis.
Authors: Junso Fujita / Fumiaki Makino / Haruyasu Asahara / Maiko Moriguchi / Shota Kumano / Itsuki Anzai / Jun-Ichi Kishikawa / Yoshiharu Matsuura / Takayuki Kato / Keiichi Namba / Tsuyoshi Inoue /
Abstract: Functionalization of graphene is one of the most important fundamental technologies in a wide variety of fields including industry and biochemistry. We have successfully achieved a novel oxidative ...Functionalization of graphene is one of the most important fundamental technologies in a wide variety of fields including industry and biochemistry. We have successfully achieved a novel oxidative modification of graphene using photoactivated ClO as a mild oxidant and confirmed the oxidized graphene grid is storable with its functionality for at least three months under N atmosphere. Subsequent chemical functionalization enabled us to develop an epoxidized graphene grid (EG-grid™), which effectively adsorbs protein particles for electron cryomicroscopy (cryoEM) image analysis. The EG-grid dramatically improved the particle density and orientation distribution. The density maps of GroEL and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were reconstructed at 1.99 and 2.16 Å resolution from only 504 and 241 micrographs, respectively. A sample solution of 0.1 mg ml was sufficient to reconstruct a 3.10 Å resolution map of SARS-CoV-2 spike protein from 1163 micrographs. The map resolutions of β-galactosidase and apoferritin easily reached 1.81 Å and 1.29 Å resolution, respectively, indicating its atomic-resolution imaging capability. Thus, the EG-grid will be an extremely powerful tool for highly efficient high-resolution cryoEM structural analysis of biological macromolecules.
History
DepositionMay 17, 2021-
Header (metadata) releaseMay 25, 2022-
Map releaseMay 25, 2022-
UpdateFeb 22, 2023-
Current statusFeb 22, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_31312.png

Downloads & links

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Map

FileDownload / File: emd_31312.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.99 Å/pix.
x 300 pix.
= 297. Å		0.99 Å/pix.
x 300 pix.
= 297. Å		0.99 Å/pix.
x 300 pix.
= 297. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.99 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.025
Minimum - Maximum-0.07947131 - 0.13799886
Average (Standard dev.)0.0002549171 (±0.003695176)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 297.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_31312_msk_1.map
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Additional map: #1

Fileemd_31312_additional_1.map
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Half map: #1

Fileemd_31312_half_map_1.map
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Half map: #2

Fileemd_31312_half_map_2.map
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Sample components

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Entire : The chimeric complex of A3B3 ring of V-ATPase from Thermus thermo...

EntireName: The chimeric complex of A3B3 ring of V-ATPase from Thermus thermophilus and DF subunit of V-ATPase from human.
Components
  • Complex: The chimeric complex of A3B3 ring of V-ATPase from Thermus thermophilus and DF subunit of V-ATPase from human.
    • Complex: The chimeric complex of A3B3 ring from Thermus thermophilus
      • Protein or peptide: A subunit of V1-ATPase from Thermus thermophilus.
    • Complex: DF subunit from human
      • Protein or peptide: B subunit of V1-ATPase from Thermus thermophilus.

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Supramolecule #1: The chimeric complex of A3B3 ring of V-ATPase from Thermus thermo...

SupramoleculeName: The chimeric complex of A3B3 ring of V-ATPase from Thermus thermophilus and DF subunit of V-ATPase from human.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 395 KDa

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Supramolecule #2: The chimeric complex of A3B3 ring from Thermus thermophilus

SupramoleculeName: The chimeric complex of A3B3 ring from Thermus thermophilus
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Thermus thermophilus (unknown)

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Supramolecule #3: DF subunit from human

SupramoleculeName: DF subunit from human / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: A subunit of V1-ATPase from Thermus thermophilus.

MacromoleculeName: A subunit of V1-ATPase from Thermus thermophilus. / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (unknown)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (unknown)
SequenceString:

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Macromolecule #2: B subunit of V1-ATPase from Thermus thermophilus.

MacromoleculeName: B subunit of V1-ATPase from Thermus thermophilus. / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (unknown)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (unknown)
SequenceString:

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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

Concentration1.0 mg/mL
BufferpH: 8
Component:
ConcentrationName
20.0 mMTris-HClTris
150.0 mMsodium chloride

Details: pH8.0
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: GRAPHENE
Details: The graphene grid was chemically oxidized and modified.
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 50000
Specialist opticsEnergy filter - Name: In-column Omega Filter / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: JEOL CRYOSPECPORTER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Average exposure time: 4.0 sec. / Average electron dose: 62.0 e/Å2

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Image processing

Particle selectionNumber selected: 1193032
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.03 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 238765
FSC plot (resolution estimation)

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