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- EMDB-13878: Cryo-electron tomogram from a cryo-FIB lift-out lamella of Drosop... -

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Entry
Database: EMDB / ID: EMD-13878
TitleCryo-electron tomogram from a cryo-FIB lift-out lamella of Drosophila melanogaster egg chambers
Map dataTomogram from cryo-lift-out of D. melanogaster egg chambers
Sample
  • Tissue: D. melanogaster
Keywordscyro-FIB / cryo-electron tomography / D. melanogaster / Golgi apparatus / RIBOSOME
Biological speciesDrosophila melanogaster (fruit fly)
Methodelectron tomography / cryo EM
AuthorsKlumpe S / Fung HKH / Goetz SK / Plitzko JM / Mahamid J
Funding supportEuropean Union, 3 items
OrganizationGrant numberCountry
EIPOD fellowship under Marie Sklodowska-Curie Actions COFUND664726European Union
European Research Council (ERC)760067European Union
European Union (EU)871037European Union
CitationJournal: Elife / Year: 2021
Title: A modular platform for automated cryo-FIB workflows.
Authors: Sven Klumpe / Herman Kh Fung / Sara K Goetz / Ievgeniia Zagoriy / Bernhard Hampoelz / Xiaojie Zhang / Philipp S Erdmann / Janina Baumbach / Christoph W Müller / Martin Beck / Jürgen M ...Authors: Sven Klumpe / Herman Kh Fung / Sara K Goetz / Ievgeniia Zagoriy / Bernhard Hampoelz / Xiaojie Zhang / Philipp S Erdmann / Janina Baumbach / Christoph W Müller / Martin Beck / Jürgen M Plitzko / Julia Mahamid /
Abstract: Lamella micromachining by focused ion beam milling at cryogenic temperature (cryo-FIB) has matured into a preparation method widely used for cellular cryo-electron tomography. Due to the limited ...Lamella micromachining by focused ion beam milling at cryogenic temperature (cryo-FIB) has matured into a preparation method widely used for cellular cryo-electron tomography. Due to the limited ablation rates of low Ga ion beam currents required to maintain the structural integrity of vitreous specimens, common preparation protocols are time-consuming and labor intensive. The improved stability of new-generation cryo-FIB instruments now enables automated operations. Here, we present an open-source software tool, SerialFIB, for creating automated and customizable cryo-FIB preparation protocols. The software encompasses a graphical user interface for easy execution of routine lamellae preparations, a scripting module compatible with available Python packages, and interfaces with three-dimensional correlative light and electron microscopy (CLEM) tools. SerialFIB enables the streamlining of advanced cryo-FIB protocols such as multi-modal imaging, CLEM-guided lamella preparation and in situ lamella lift-out procedures. Our software therefore provides a foundation for further development of advanced cryogenic imaging and sample preparation protocols.
History
DepositionNov 15, 2021-
Header (metadata) releaseJan 19, 2022-
Map releaseJan 19, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
  • Download
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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Movie viewer
Supplemental images

emd_13878.png

Downloads & links

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Map

FileDownload / File: emd_13878.map.gz / Format: CCP4 / Size: 768.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTomogram from cryo-lift-out of D. melanogaster egg chambers
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
14.08 Å/pix.
x 234 pix.
= 3294.72 Å		14.08 Å/pix.
x 928 pix.
= 13066.24 Å		14.08 Å/pix.
x 928 pix.
= 13066.24 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 14.08 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-69.493645000000001 - 88.434139999999999
Average (Standard dev.)1.2848316 (±7.7204328)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin928928117
Dimensions928928234
Spacing928928234
CellA: 13066.24 Å / B: 13066.24 Å / C: 3294.72 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z14.0814.0814.08
M x/y/z928928234
origin x/y/z0.0000.0000.000
length x/y/z13066.24013066.2403294.720
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ360360360
MAP C/R/S123
start NC/NR/NS928928117
NC/NR/NS928928234
D min/max/mean-69.49488.4341.285

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Supplemental data

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Sample components

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Entire : D. melanogaster

EntireName: D. melanogaster
Components
  • Tissue: D. melanogaster

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Supramolecule #1: D. melanogaster

SupramoleculeName: D. melanogaster / type: tissue / ID: 1 / Parent: 0
Details: Tomogram from cryo-FIB lift-out lamella performed on a D. melanogaster egg chamber
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Strain: w[*]; P{w[+mC]=sqh-mCherry.M}3 (Flybase ID: FBst0059024)
Tissue: Egg Chamber

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation statetissue

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: NITROGEN / Details: Leica EM Ice.
High pressure freezingInstrument: OTHER
Details: 20% ficol in Schneider's medium. The value given for _em_high_pressure_freezing.instrument is EM Ice. This is not in a list of allowed values {'LEICA EM HPM100', 'LEICA EM PACT', 'BAL-TEC ...Details: 20% ficol in Schneider's medium. The value given for _em_high_pressure_freezing.instrument is EM Ice. This is not in a list of allowed values {'LEICA EM HPM100', 'LEICA EM PACT', 'BAL-TEC HPM 010', 'LEICA EM PACT2', 'EMS-002 RAPID IMMERSION FREEZER', 'OTHER'} so OTHER is written into the XML file.
Cryo protectantFicol
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 / Focused ion beam - Current: 1 / Focused ion beam - Duration: 60 / Focused ion beam - Temperature: 93 K / Focused ion beam - Initial thickness: 10000 / Focused ion beam - Final thickness: 200
Focused ion beam - Details: Cryo-FIB Lift-Out method Various milling steps. The value given for _em_focused_ion_beam.instrument is TFS Aquilos. This is not in a list of allowed values {'DB235', ...Focused ion beam - Details: Cryo-FIB Lift-Out method Various milling steps. The value given for _em_focused_ion_beam.instrument is TFS 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

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Average electron dose: 2.0 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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionSoftware - Name: IMOD / Number images used: 59

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