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- EMDB-16136: Cryo-electron tomogram acquired on a cryo-FIB lamella of a retina... -

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Basic information

Entry
Database: EMDB / ID: EMD-16136
TitleCryo-electron tomogram acquired on a cryo-FIB lamella of a retinal pigment epithelial (RPE1) cell
Map dataReconstructed cryo-electron tomogram acquired on RPE1 cryo-FIB lamella
Sample
  • Organelle or cellular component: RPE1 cytosol with actin stress fiber
KeywordsActin stress fiber / CYTOSOLIC PROTEIN
Biological speciesHomo sapiens (human)
Methodelectron tomography / cryo EM
AuthorsMahamid J / Goetz SK
Funding supportEuropean Union, 1 items
OrganizationGrant numberCountry
European Research Council (ERC)760067European Union
CitationJournal: Nat Methods / Year: 2020
Title: Tailoring cryo-electron microscopy grids by photo-micropatterning for in-cell structural studies.
Authors: Mauricio Toro-Nahuelpan / Ievgeniia Zagoriy / Fabrice Senger / Laurent Blanchoin / Manuel Théry / Julia Mahamid /
Abstract: Spatially controlled cell adhesion on electron microscopy supports remains a bottleneck in specimen preparation for cellular cryo-electron tomography. Here, we describe contactless and mask-free ...Spatially controlled cell adhesion on electron microscopy supports remains a bottleneck in specimen preparation for cellular cryo-electron tomography. Here, we describe contactless and mask-free photo-micropatterning of electron microscopy grids for site-specific deposition of extracellular matrix-related proteins. We attained refined cell positioning for micromachining by cryo-focused ion beam milling. Complex micropatterns generated predictable intracellular organization, allowing direct correlation between cell architecture and in-cell three-dimensional structural characterization of the underlying molecular machinery.
History
DepositionNov 14, 2022-
Header (metadata) releaseNov 23, 2022-
Map releaseNov 23, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16136.png

Downloads & links

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Map

FileDownload / File: emd_16136.map.gz / Format: CCP4 / Size: 513.3 MB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationReconstructed cryo-electron tomogram acquired on RPE1 cryo-FIB lamella
Voxel sizeX=Y=Z: 13.481 Å
Density
Minimum - Maximum-128.0 - 127.0
Average (Standard dev.)-1.4660258 (±27.244865000000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00313
Dimensions928928625
Spacing928928625
CellA: 12510.368 Å / B: 12510.368 Å / C: 8425.625 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16136_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : RPE1 cytosol with actin stress fiber

EntireName: RPE1 cytosol with actin stress fiber
Components
  • Organelle or cellular component: RPE1 cytosol with actin stress fiber

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Supramolecule #1: RPE1 cytosol with actin stress fiber

SupramoleculeName: RPE1 cytosol with actin stress fiber / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human) / Strain: RPE1 cell line / Location in cell: cytosol

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7.4
GridModel: Quantifoil / Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE
Cryo protectantNone
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 / Focused ion beam - Current: 0.05 / Focused ion beam - Duration: 120 / Focused ion beam - Temperature: 88 K / Focused ion beam - Initial thickness: 1000 / Focused ion beam - Final thickness: 200
Focused ion beam - Details: The value given for _em_focused_ion_beam.instrument is Thermo Fisher Aquilos. This is not in a list of allowed values {'DB235', 'OTHER'} so OTHER is written into the XML file.

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 42000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 4.0 µm / Nominal defocus min: 4.0 µm
Specialist opticsPhase plate: VOLTA PHASE PLATE / Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 2.2 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: IMOD / Number images used: 57

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