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- EMDB-34106: GroEL on EG-grid stored for 3 months after graphene oxidation -

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Basic information

Entry
Database: EMDB / ID: EMD-34106
TitleGroEL on EG-grid stored for 3 months after graphene oxidation
Map data
Sample
  • Complex: GroEL
    • Protein or peptide: GroEL
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.06 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
DepositionAug 17, 2022-
Header (metadata) releaseFeb 22, 2023-
Map releaseFeb 22, 2023-
UpdateFeb 22, 2023-
Current statusFeb 22, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_34106.png

Downloads & links

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Map

FileDownload / File: emd_34106.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.868 Å
Density
Contour LevelBy AUTHOR: 0.0081
Minimum - Maximum-0.04891688 - 0.097418495
Average (Standard dev.)0.0001485362 (±0.0025701148)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 312.47998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_34106_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Filtered map

Fileemd_34106_additional_1.map
AnnotationFiltered map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_34106_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_34106_half_map_2.map
Projections & Slices
AxesZYX

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Sample components

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Entire : GroEL

EntireName: GroEL
Components
  • Complex: GroEL
    • Protein or peptide: GroEL

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Supramolecule #1: GroEL

SupramoleculeName: GroEL / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Details: The sample was purchased from TaKaRa Bio Inc. (Product code 7330).
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 802 KDa

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Macromolecule #1: GroEL

MacromoleculeName: GroEL / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
SequenceString: MAAKDVKFGN DARVKMLRGV NVLADAVKVT LGPKGRNVVL DKSFGAPTIT KDGVSVAREI ELEDKFENM GAQMVKEVAS KANDAAGDGT TTATVLAQAI ITEGLKAVAA GMNPMDLKRG I DKAVTAAV EELKALSVPC SDSKAIAQVG TISANSDETV GKLIAEAMDK ...String:
MAAKDVKFGN DARVKMLRGV NVLADAVKVT LGPKGRNVVL DKSFGAPTIT KDGVSVAREI ELEDKFENM GAQMVKEVAS KANDAAGDGT TTATVLAQAI ITEGLKAVAA GMNPMDLKRG I DKAVTAAV EELKALSVPC SDSKAIAQVG TISANSDETV GKLIAEAMDK VGKEGVITVE DG TGLQDEL DVVEGMQFDR GYLSPYFINK PETGAVELES PFILLADKKI SNIREMLPVL EAV AKAGKP LLIIAEDVEG EALATLVVNT MRGIVKVAAV KAPGFGDRRK AMLQDIATLT GGTV ISEEI GMELEKATLE DLGQAKRVVI NKDTTTIIDG VGEEAAIQGR VAQIRQQIEE ATSDY DREK LQERVAKLAG GVAVIKVGAA TEVEMKEKKA RVEDALHATR AAVEEGVVAG GGVALI RVA SKLADLRGQN EDQNVGIKVA LRAMEAPLRQ IVLNCGEEPS VVANTVKGGD GNYGYNA AT EEYGNMIDMG ILDPTKVTRS ALQYAASVAG LMITTECMVT DLPKNDAADL GAAGGMGG M GGMGGMM

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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

Concentration1.5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
25.0 mMHEPES2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid
50.0 mMNaClSodium chloridesodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: GRAPHENE
Details: The graphene grid was oxidized and stored under nitrogen gas at room temperature for three months. Then the grid was chemically 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: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 60000
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: 2.6 sec. / Average electron dose: 40.0 e/Å2

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Image processing

Particle selectionNumber selected: 154796
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.0)
Final reconstructionApplied symmetry - Point group: D7 (2x7 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.06 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0) / Number images used: 52043
FSC plot (resolution estimation)

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