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- EMDB-13834: Subtomogram average of 80S ribosomes from a cryo-FIB-lamella of S... -

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Entry
Database: EMDB / ID: EMD-13834
TitleSubtomogram average of 80S ribosomes from a cryo-FIB-lamella of Sum159 human cell line prepared after cryo-FIB-SEM volume imaging
Map dataSubtomogram average of Sum159 ribosomes from 2 tomograms (TS_001.mrc, TS_005.mrc) obtained from a cryo-FIB-milled lamella after cryo-FIB-SEM volume imaging
Sample
  • Complex: 80S ribosomeEukaryotic ribosome
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 24.0 Å
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
  • Surface view with section colored by density value
  • Surface level: 0.15
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.15
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13834.png

Downloads & links

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Map

FileDownload / File: emd_13834.map.gz / Format: CCP4 / Size: 10.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomogram average of Sum159 ribosomes from 2 tomograms (TS_001.mrc, TS_005.mrc) obtained from a cryo-FIB-milled lamella after cryo-FIB-SEM volume imaging
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.37 Å/pix.
x 140 pix.
= 471.828 Å		3.37 Å/pix.
x 140 pix.
= 471.828 Å		3.37 Å/pix.
x 140 pix.
= 471.828 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.3702 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15 / Movie #1: 0.15
Minimum - Maximum-0.20402384 - 0.41610524
Average (Standard dev.)0.0056422907 (±0.055197176)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions140140140
Spacing140140140
CellA=B=C: 471.828 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.37023.37023.3702
M x/y/z140140140
origin x/y/z0.0000.0000.000
length x/y/z471.828471.828471.828
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS140140140
D min/max/mean-0.2040.4160.006

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Supplemental data

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Sample components

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Entire : 80S ribosome

EntireName: 80S ribosomeEukaryotic ribosome
Components
  • Complex: 80S ribosomeEukaryotic ribosome

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Supramolecule #1: 80S ribosome

SupramoleculeName: 80S ribosome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human) / Strain: Sum159 / Location in cell: cytoplasm

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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: ETHANE

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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: 2.27915 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 2 / Number images used: 3380
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 24.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number subtomograms used: 3380
FSC plot (resolution estimation)

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