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- EMDB-32159: GroEL on hydrophilized graphene grid (high particle density) -

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Basic information

Entry
Database: EMDB / ID: EMD-32159
TitleGroEL on hydrophilized graphene grid (high particle density)
Map data
Sample
  • Complex: GroEL
    • Protein or peptide: GroEL
KeywordsGroEL / 60 kDa chaperonin / CHAPERONE
Function / homology
Function and homology information


GroEL-GroES complex / chaperonin ATPase / virion assembly / chaperone cofactor-dependent protein refolding / isomerase activity / ATP-dependent protein folding chaperone / response to radiation / unfolded protein binding / protein folding / response to heat ...GroEL-GroES complex / chaperonin ATPase / virion assembly / chaperone cofactor-dependent protein refolding / isomerase activity / ATP-dependent protein folding chaperone / response to radiation / unfolded protein binding / protein folding / response to heat / protein refolding / magnesium ion binding / ATP hydrolysis activity / ATP binding / membrane / identical protein binding / cytosol
Similarity search - Function
Chaperonin Cpn60, conserved site / Chaperonins cpn60 signature. / Chaperonin Cpn60/GroEL / GroEL-like equatorial domain superfamily / TCP-1-like chaperonin intermediate domain superfamily / GroEL-like apical domain superfamily / TCP-1/cpn60 chaperonin family / Chaperonin Cpn60/GroEL/TCP-1 family
Similarity search - Domain/homology
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.09 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
DepositionNov 11, 2021-
Header (metadata) releaseNov 16, 2022-
Map releaseNov 16, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_32159.png

Downloads & links

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Map

FileDownload / File: emd_32159.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.878 Å
Density
Contour LevelBy AUTHOR: 0.017
Minimum - Maximum-0.11115054 - 0.20599538
Average (Standard dev.)0.000265012 (±0.0054001594)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 316.08002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_32159_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Locally filtered map

Fileemd_32159_additional_1.map
AnnotationLocally filtered map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_32159_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_32159_half_map_2.map
Projections & Slices
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Histogram (log scale)

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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 / 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 ELEDKFENMG AQMVKEVASK ANDAAGDGTT TATVLAQAII TEGLKAVAAG MNPMDLKRGI DKAVTAAVEE LKALSVPCSD SKAIAQVGTI SANSDETVGK LIAEAMDKVG ...String:
MAAKDVKFGN DARVKMLRGV NVLADAVKVT LGPKGRNVVL DKSFGAPTIT KDGVSVAREI ELEDKFENMG AQMVKEVASK ANDAAGDGTT TATVLAQAII TEGLKAVAAG MNPMDLKRGI DKAVTAAVEE LKALSVPCSD SKAIAQVGTI SANSDETVGK LIAEAMDKVG KEGVITVEDG TGLQDELDVV EGMQFDRGYL SPYFINKPET GAVELESPFI LLADKKISNI REMLPVLEAV AKAGKPLLII AEDVEGEALA TLVVNTMRGI VKVAAVKAPG FGDRRKAMLQ DIATLTGGTV ISEEIGMELE KATLEDLGQA KRVVINKDTT TIIDGVGEEA AIQGRVAQIR QQIEEATSDY DREKLQERVA KLAGGVAVIK VGAATEVEMK EKKARVEDAL HATRAAVEEG VVAGGGVALI RVASKLADLR GQNEDQNVGI KVALRAMEAP LRQIVLNCGE EPSVVANTVK GGDGNYGYNA ATEEYGNMID MGILDPTKVT RSALQYAASV AGLMITTECM VTDLPKNDAA DLGAAGGMGG MGGMGGMM

UniProtKB: Chaperonin GroEL

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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:
ConcentrationFormula
25.0 mMHEPES
50.0 mMNaClSodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: GRAPHENE / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
Details: To avoid graphene breakage, a loose mask made of a thin aluminum foil was placed above the grids during the glow discharge.
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: 2.8 sec. / Average electron dose: 40.0 e/Å2

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Image processing

Particle selectionNumber selected: 340446
Startup modelType of model: NONE
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: D7 (2x7 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.09 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 198677
FSC plot (resolution estimation)

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