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- EMDB-50702: E. coli 70S ribosome in situ structure for lamellae backside dama... -

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Basic information

Entry
Database: EMDB / ID: EMD-50702
TitleE. coli 70S ribosome in situ structure for lamellae backside damage analysis: 3 - 4 microns from lamellae backside
Map data
Sample
  • Cell: E. coli 70S ribosome
Keywords70S E. coli ribosome / RIBOSOME
Biological speciesEscherichia coli (E. coli)
Methodsubtomogram averaging / cryo EM / Resolution: 6.6 Å
AuthorsWatson H / Berger C / Grange M
Funding support United Kingdom, 3 items
OrganizationGrant numberCountry
UK Research and Innovation (UKRI) United Kingdom
Wellcome Trust220526/Z/20/Z United Kingdom
Wellcome Trust225902/Z/22/Z United Kingdom
CitationJournal: Nat Commun / Year: 2025
Title: Xenon plasma focused ion beam lamella fabrication on high-pressure frozen specimens for structural cell biology.
Authors: Casper Berger / Helena Watson / James H Naismith / Maud Dumoux / Michael Grange /
Abstract: Cryo focused ion beam lamella preparation is a potent tool for in situ structural biology, enabling the study of macromolecules in their native cellular environments. However, throughput is currently ...Cryo focused ion beam lamella preparation is a potent tool for in situ structural biology, enabling the study of macromolecules in their native cellular environments. However, throughput is currently limited, especially for thicker, more biologically complex samples. We describe how xenon plasma focused ion beam milling can be used for routine bulk milling of thicker, high-pressure frozen samples. We demonstrate lamellae preparation with a high success rate on these samples and determine a 4.0 Å structure of the Escherichia coli ribosome on these lamellae using sub volume averaging. We determine the effects on sample integrity of increased ion currents up to 60 nA during bulk milling of thicker planar samples, showing no measurable damage to macromolecules beyond an amorphous layer on the backside of the lamellae. The use of xenon results in substantial structural damage to particles up to approximately 30 nm in depth from the milled surfaces, and the effects of damage become negligibly small by 45 nm. Our results outline how the use of high currents using xenon plasma focused ion beam milling may be integrated into FIB milling regimes for preparing thin lamellae for high-resolution in situ structural biology.
History
DepositionJun 18, 2024-
Header (metadata) releaseMar 19, 2025-
Map releaseMar 19, 2025-
UpdateMar 19, 2025-
Current statusMar 19, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50702.png

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Map

FileDownload / File: emd_50702.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.9 Å/pix.
x 224 pix.
= 425.6 Å		1.9 Å/pix.
x 224 pix.
= 425.6 Å		1.9 Å/pix.
x 224 pix.
= 425.6 Å

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.9 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.627
Minimum - Maximum-0.86303306 - 2.0988374
Average (Standard dev.)0.012307618 (±0.18171614)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 425.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_50702_msk_1.map
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Half map: #1

Fileemd_50702_half_map_1.map
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Half map: #2

Fileemd_50702_half_map_2.map
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Sample components

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Entire : E. coli 70S ribosome

EntireName: E. coli 70S ribosome
Components
  • Cell: E. coli 70S ribosome

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Supramolecule #1: E. coli 70S ribosome

SupramoleculeName: E. coli 70S ribosome / type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli (E. coli) / Strain: C43 (DE3)

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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: 7
VitrificationCryogen name: NITROGEN / Details: Leica EM ICE HPF.
DetailsHigh-pressure frozen E. coli milled with xenon plasma ion source

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number real images: 1 / Average electron dose: 5.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 64000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0) / Number subtomograms used: 7623
ExtractionNumber tomograms: 167 / Number images used: 224823 / Method: crYOLO / Software: (Name: Warp (ver. 1.0.9), crYOLO (ver. 1.8))
Final angle assignmentType: OTHER / Software - Name: RELION (ver. 4.0)
FSC plot (resolution estimation)

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