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- EMDB-16199: 80S human ribosome structure from PFIB lamellae of HeLa cells for... -

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Basic information

Entry
Database: EMDB / ID: EMD-16199
Title80S human ribosome structure from PFIB lamellae of HeLa cells for assessing the extend and depth of the damage layer: above 15 nm matched control (for 0 to 15 nm)
Map data
Sample
  • Cell: 80S human ribosome
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 13.2 Å
AuthorsBerger C / Grange M
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust220526/Z/20/Z United Kingdom
CitationJournal: Nat Commun / Year: 2023
Title: Plasma FIB milling for the determination of structures in situ.
Authors: Casper Berger / Maud Dumoux / Thomas Glen / Neville B-Y Yee / John M Mitchels / Zuzana Patáková / Michele C Darrow / James H Naismith / Michael Grange /
Abstract: Structural biology studies inside cells and tissues require methods to thin vitrified specimens to electron transparency. Until now, focused ion beams based on gallium have been used. However, ion ...Structural biology studies inside cells and tissues require methods to thin vitrified specimens to electron transparency. Until now, focused ion beams based on gallium have been used. However, ion implantation, changes to surface chemistry and an inability to access high currents limit gallium application. Here, we show that plasma-coupled ion sources can produce cryogenic lamellae of vitrified human cells in a robust and automated manner, with quality sufficient for pseudo-atomic structure determination. Lamellae were produced in a prototype microscope equipped for long cryogenic run times (> 1 week) and with multi-specimen support fully compatible with modern-day transmission electron microscopes. We demonstrate that plasma ion sources can be used for structural biology within cells, determining a structure in situ to 4.9 Å, and characterise the resolution dependence on particle distance from the lamella edge. We describe a workflow upon which different plasmas can be examined to further streamline lamella fabrication.
History
DepositionNov 22, 2022-
Header (metadata) releaseJan 25, 2023-
Map releaseJan 25, 2023-
UpdateFeb 15, 2023-
Current statusFeb 15, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16199.png

Downloads & links

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Map

FileDownload / File: emd_16199.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
1.9 Å/pix.
x 180 pix.
= 342. Å		1.9 Å/pix.
x 180 pix.
= 342. Å		1.9 Å/pix.
x 180 pix.
= 342. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.9 Å
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Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-0.51261836 - 1.6423408
Average (Standard dev.)0.094904296 (±0.22151528)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 342.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16199_msk_1.map
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Additional map: #1

Fileemd_16199_additional_1.map
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Additional map: #2

Fileemd_16199_additional_2.map
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Half map: #1

Fileemd_16199_half_map_1.map
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Half map: #2

Fileemd_16199_half_map_2.map
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Sample components

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Entire : 80S human ribosome

EntireName: 80S human ribosome
Components
  • Cell: 80S human ribosome

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Supramolecule #1: 80S human ribosome

SupramoleculeName: 80S human ribosome / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 5.75
GridModel: UltrAuFoil R2/2 / Material: GOLD / Mesh: 200 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV
Details: 24 h post infection cells were plunge-frozen using the Vitrobot (Thermo Fisher) offsetting the blotting pad to favour back blotting. Just prior the PF, the cells media has been replaced with ...Details: 24 h post infection cells were plunge-frozen using the Vitrobot (Thermo Fisher) offsetting the blotting pad to favour back blotting. Just prior the PF, the cells media has been replaced with the complete media with 10% glycerol (v/v)..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 64000
Specialist opticsEnergy filter - Name: TFS Selectris / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 2 / Average electron dose: 5.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 180 / Number images used: 70436 / Method: 3D classification
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 13.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Warp (ver. 1.09) / Software - details: Warp/M / Number subtomograms used: 351
FSC plot (resolution estimation)

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