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- EMDB-18028: Charging of vitreous samples in cryogenic electron microscopy mit... -

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Entry
Database: EMDB / ID: EMD-18028
TitleCharging of vitreous samples in cryogenic electron microscopy mitigated by graphene - BfrB - Dataset 2 - Quantifoil 300 mesh R1.2/1.3 with Graphene - Small Beam
Map data
Sample
  • Complex: MYCOBACTERIUM TUBERCULOSIS FERRITIN
    • Protein or peptide: Ferritin BfrB
KeywordsIRON STORAGE / FERROXIDASE / BACTERIAL FERRITIN / OCTAHEDRAL SYMMETRY. / METAL TRANSPORT / METAL BINDING PROTEIN
Biological speciesMycobacterium tuberculosis H37Rv (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.01 Å
Authorsvan schayck JP / Zhang Y / Ravelli RBG
Funding support Netherlands, European Union, 3 items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)184.034.014 Netherlands
Health-HollandLSHM21029 Netherlands
European Commission815128European Union
CitationJournal: ACS Nano / Year: 2023
Title: Charging of Vitreous Samples in Cryogenic Electron Microscopy Mitigated by Graphene.
Authors: Yue Zhang / J Paul van Schayck / Adrián Pedrazo-Tardajos / Nathalie Claes / Willem E M Noteborn / Peng-Han Lu / Hans Duimel / Rafal E Dunin-Borkowski / Sara Bals / Peter J Peters / Raimond B G Ravelli /
Abstract: Cryogenic electron microscopy can provide high-resolution reconstructions of macromolecules embedded in a thin layer of ice from which atomic models can be built . However, the interaction between ...Cryogenic electron microscopy can provide high-resolution reconstructions of macromolecules embedded in a thin layer of ice from which atomic models can be built . However, the interaction between the ionizing electron beam and the sample results in beam-induced motion and image distortion, which limit the attainable resolutions. Sample charging is one contributing factor of beam-induced motions and image distortions, which is normally alleviated by including part of the supporting conducting film within the beam-exposed region. However, routine data collection schemes avoid strategies whereby the beam is not in contact with the supporting film, whose rationale is not fully understood. Here we characterize electrostatic charging of vitreous samples, both in imaging and in diffraction mode. We mitigate sample charging by depositing a single layer of conductive graphene on top of regular EM grids. We obtained high-resolution single-particle analysis (SPA) reconstructions at 2 Å when the electron beam only irradiates the middle of the hole on graphene-coated grids, using data collection schemes that previously failed to produce sub 3 Å reconstructions without the graphene layer. We also observe that the SPA data obtained with the graphene-coated grids exhibit a higher factor and reduced particle movement compared to data obtained without the graphene layer. This mitigation of charging could have broad implications for various EM techniques, including SPA and cryotomography, and for the study of radiation damage and the development of future sample carriers. Furthermore, it may facilitate the exploration of more dose-efficient, scanning transmission EM based SPA techniques.
History
DepositionJul 26, 2023-
Header (metadata) releaseAug 2, 2023-
Map releaseAug 2, 2023-
UpdateAug 30, 2023-
Current statusAug 30, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18028.png

Downloads & links

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Map

FileDownload / File: emd_18028.map.gz / Format: CCP4 / Size: 129.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.834 Å
Density
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.04146692 - 0.09296244
Average (Standard dev.)0.000003987217 (±0.004581295)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions324324324
Spacing324324324
CellA=B=C: 270.216 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_18028_half_map_1.map
Projections & Slices
AxesZYX

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Half map: #1

Fileemd_18028_half_map_2.map
Projections & Slices
AxesZYX

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Sample components

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Entire : MYCOBACTERIUM TUBERCULOSIS FERRITIN

EntireName: MYCOBACTERIUM TUBERCULOSIS FERRITIN
Components
  • Complex: MYCOBACTERIUM TUBERCULOSIS FERRITIN
    • Protein or peptide: Ferritin BfrB

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Supramolecule #1: MYCOBACTERIUM TUBERCULOSIS FERRITIN

SupramoleculeName: MYCOBACTERIUM TUBERCULOSIS FERRITIN / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: MYCOBACTERIUM TUBERCULOSIS FERRITIN
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)

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Macromolecule #1: Ferritin BfrB

MacromoleculeName: Ferritin BfrB / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: ferroxidase
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MTEYEGPKTK FHALMQEQI H NEFTAAQQ YV AIAVYFD SED LPQLAK HFYS QAVEE RNHAM MLVQ HLLDRD LRV EIPGVDT VR NQFDRPRE A LALALDQER TVTDQVGRLT AVARDEGDF L GEQFMQWF LQ EQIEEVA LMA TLVRVA DRAG ANLFE ...String:
MTEYEGPKTK FHALMQEQI H NEFTAAQQ YV AIAVYFD SED LPQLAK HFYS QAVEE RNHAM MLVQ HLLDRD LRV EIPGVDT VR NQFDRPRE A LALALDQER TVTDQVGRLT AVARDEGDF L GEQFMQWF LQ EQIEEVA LMA TLVRVA DRAG ANLFE LENFV AREV DVAPAA SGA PHAAGGR L

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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

Concentration5 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
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 / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 6000 pixel / Digitization - Dimensions - Height: 4000 pixel / Number grids imaged: 1 / Number real images: 621 / Average exposure time: 1.7 sec. / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 146626
Startup modelType of model: NONE
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: O (octahedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.01 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0) / Number images used: 146626
FSC plot (resolution estimation)

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