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- EMDB-13833: Cryo-electron tomograms from cryo-FIB-lamellae of Sum159 human ce... -

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Basic information

Entry
Database: EMDB / ID: EMD-13833
TitleCryo-electron tomograms from cryo-FIB-lamellae of Sum159 human cell line
Map dataTomogram obtained from a cryo-FIB-milled Sum159 lamella
Sample
  • Cell: Sum159
Biological speciesHomo sapiens (human)
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 3, 2021-
Header (metadata) releaseJan 12, 2022-
Map releaseJan 12, 2022-
UpdateJan 12, 2022-
Current statusJan 12, 2022Processing 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
  • Download
Movie viewer
Supplemental images

emd_13833.png

Downloads & links

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Map

FileDownload / File: emd_13833.map.gz / Format: CCP4 / Size: 1.7 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTomogram obtained from a cryo-FIB-milled Sum159 lamella
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
13.48 Å/pix.
x 500 pix.
= 6740.4 Å		13.48 Å/pix.
x 928 pix.
= 12510.182 Å		13.48 Å/pix.
x 960 pix.
= 12941.567 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: 13.4808 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-103.03785 - 98.147095
Average (Standard dev.)0.5209236 (±4.4466887)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-1616250
Dimensions928960500
Spacing960928500
CellA: 12941.567 Å / B: 12510.182 Å / C: 6740.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z13.48079895833313.48079956896613.4808
M x/y/z960928500
origin x/y/z0.0000.0000.000
length x/y/z12941.56712510.1826740.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS16-16250
NC/NR/NS960928500
D min/max/mean-103.03898.1470.521

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Supplemental data

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Additional map: Tomogram obtained from a cryo-FIB-milled Sum159 lamella used...

Fileemd_13833_additional_1.map
AnnotationTomogram obtained from a cryo-FIB-milled Sum159 lamella used for subtomogram averaging
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Tomogram obtained from a cryo-FIB-milled Sum159 lamella used...

Fileemd_13833_additional_2.map
AnnotationTomogram obtained from a cryo-FIB-milled Sum159 lamella used for subtomogram averaging
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Tomogram obtained from a cryo-FIB-milled Sum159 lamella used...

Fileemd_13833_additional_3.map
AnnotationTomogram obtained from a cryo-FIB-milled Sum159 lamella used for subtomogram averaging
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Tomogram obtained from a cryo-FIB-milled Sum159 lamella used...

Fileemd_13833_additional_4.map
AnnotationTomogram obtained from a cryo-FIB-milled Sum159 lamella used for subtomogram averaging
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Sum159

EntireName: Sum159
Components
  • Cell: Sum159

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Supramolecule #1: Sum159

SupramoleculeName: Sum159 / type: cell / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human) / Strain: Sum159

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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
VitrificationCryogen name: ETHANE
SectioningFocused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 kV / Focused ion beam - Current: 0.05 nA / Focused ion beam - Duration: 150 sec. / Focused ion beam - Temperature: 88 K / Focused ion beam - Initial thickness: 1000 nm / Focused ion beam - Final thickness: 200 nm
Focused ion beam - Details: The value given for _emd_sectioning_focused_ion_beam.instrument is Thermo Fisher Aquilos. This is not in a list of allowed values {'OTHER', 'DB235'} so OTHER is written into the XML file.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
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 QUANTUM (4k x 4k) / Detector mode: COUNTING / Average electron dose: 2.29246 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: 58

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