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- EMDB-30399: Escherichia coli 70S Ribosome Reconstruction from Graphene Oxide ... -

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Basic information

Entry
Database: EMDB / ID: EMD-30399
TitleEscherichia coli 70S Ribosome Reconstruction from Graphene Oxide Monolayer fabricated Holey Carbon grid
Map data
Sample
  • Complex: 3D Reconstruction of Escherichia coli 70S Ribosome Vitrified on Graphene Oxide Monolayer fabricated Holey Carbon grid
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsKumar A / Sengupta N / Dutta S
Funding support India, 5 items
OrganizationGrant numberCountry
Department of Science & Technology (DST, India)SB/S2/RJN-145/2015 India
Science and Engineering Research Board (SERB)SERB-EMR/2016/000608) India
Department of Biotechnology (DBT, India)BT/PR25580/BRB/10/1619/2017) India
Department of Biotechnology (DBT, India)BT/INF/22/SP22844/2017 India
Department of Science & Technology (DST, India)SR/FST/LSII-039/2015 India
CitationJournal: Nanomaterials (Basel) / Year: 2021
Title: Simplified Approach for Preparing Graphene Oxide TEM Grids for Stained and Vitrified Biomolecules.
Authors: Anil Kumar / Nayanika Sengupta / Somnath Dutta /
Abstract: In this manuscript, we report the application of graphene oxide (GO) in the preparation of cryo-electron microscopy (cryo-EM) and transmission electron microscopy (TEM) grids. We treated GO with ...In this manuscript, we report the application of graphene oxide (GO) in the preparation of cryo-electron microscopy (cryo-EM) and transmission electron microscopy (TEM) grids. We treated GO with water and organic solvents, such as, methanol, ethanol and isopropanol separately to isolate significantly large GO monolayer flake to fabricate the grids for cryo-EM and TEM study. We implemented a simplified approach to isolate flakes of GO monolayer for constructing the TEM grids, independent of expensive heavy equipment (Langmuir-Blodgett trough, glow-discharge system, carbon-evaporator or plasma-cleaner or peristaltic pumps). We employed confocal microscopy, SEM and TEM to characterize the flake size, stability and transparency of the GO monolayer and atomic force microscopy (AFM) to probe the depth of GO coated grids. Additionally, GO grids are visualized at cryogenic condition for suitability of GO monolayer for cryo-EM study. In addition, GO-Met-HO grids reduce the effect of preferred orientation of biological macromolecules within the amorphous ice. The power-spectrum and contrast-transfer-function unequivocally suggest that GO-Met-HO fabricated holey grids have excellent potential for application in high-resolution structural characterization of biomolecules. Furthermore, only 200 movies and ~8000 70S ribosome particles are selected on GO-coated grids for cryo-EM reconstruction to achieve high-resolution structure.
History
DepositionJul 22, 2020-
Header (metadata) releaseMar 10, 2021-
Map releaseMar 10, 2021-
UpdateSep 21, 2022-
Current statusSep 21, 2022Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.5
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 2.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30399.png

Downloads & links

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Map

FileDownload / File: emd_30399.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.2 Å
Density
Contour LevelBy AUTHOR: 2.1 / Movie #1: 2.5
Minimum - Maximum-6.077897 - 14.206609
Average (Standard dev.)6.3472587e-12 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 360.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.21.21.2
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z360.000360.000360.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-6.07814.2070.000

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Supplemental data

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Sample components

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Entire : 3D Reconstruction of Escherichia coli 70S Ribosome Vitrified on G...

EntireName: 3D Reconstruction of Escherichia coli 70S Ribosome Vitrified on Graphene Oxide Monolayer fabricated Holey Carbon grid
Components
  • Complex: 3D Reconstruction of Escherichia coli 70S Ribosome Vitrified on Graphene Oxide Monolayer fabricated Holey Carbon grid

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Supramolecule #1: 3D Reconstruction of Escherichia coli 70S Ribosome Vitrified on G...

SupramoleculeName: 3D Reconstruction of Escherichia coli 70S Ribosome Vitrified on Graphene Oxide Monolayer fabricated Holey Carbon grid
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli B (bacteria)

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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: 7.6
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 7748
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)

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