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- EMDB-3418: Subtomogram average of 80S ribosomes obtained using the VPP -

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Basic information

Entry
Database: EMDB / ID: EMD-3418
TitleSubtomogram average of 80S ribosomes obtained using the VPP
Map dataSubtomogram average of the 80S ribosome obtained with the VPP
Sample
  • Sample: 80S ribosome
  • Complex: 80S ribosome
KeywordsRibosome / cryo-electron tomography / subtomogram averaging / phase plate
Biological speciesOryctolagus cuniculus (rabbit)
Methodsubtomogram averaging / cryo EM / Resolution: 9.6 Å
AuthorsKhoshouei M / Pfeffer S / Baumeister W / Foerster F / Danev R
CitationJournal: J Struct Biol / Year: 2017
Title: Subtomogram analysis using the Volta phase plate.
Authors: Maryam Khoshouei / Stefan Pfeffer / Wolfgang Baumeister / Friedrich Förster / Radostin Danev /
Abstract: Cryo-electron tomography (CET) and subtomogram analysis allow studying the structures of macromolecular complexes in their natural context. The radiation sensitivity of vitrified biological specimens ...Cryo-electron tomography (CET) and subtomogram analysis allow studying the structures of macromolecular complexes in their natural context. The radiation sensitivity of vitrified biological specimens and the resulting low signal-to-noise ratio (SNR) in CET limit the amount of structural information that can be mined from tomographic data. The Volta phase plate (VPP) has emerged as an effective means to increase the SNR and hence contrast compared to 'conventional' defocus-based phase contrast transmission electron microscopy (CTEM). Here, we assess the performance of the VPP compared to CTEM in subtomogram analysis, using the mammalian 80S ribosome as a test case. Accurate focusing is the major factor for achieving high resolution with the VPP, as highlighted by a comparison of slightly different focusing strategies. From only 1400 subtomograms, the VPP yields a subtomogram average of the mammalian 80S ribosome at 9.6Å resolution without laborious contrast transfer function (CTF) correction. The subtomogram averages obtained using CTEM approaches are comparable, but suffer from lower signal transfer in certain frequency bands due to the oscillations of the CTF. Our study demonstrates that the VPP is a valuable tool for subtomogram analysis, because it enables improved performance and efficiency in terms of structure localization and number of subtomograms required for a given resolution.
History
DepositionApr 29, 2016-
Header (metadata) releaseMay 18, 2016-
Map releaseJun 8, 2016-
UpdateJul 26, 2017-
Current statusJul 26, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.24
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 3.24
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3418.jpg

Downloads & links

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Map

FileDownload / File: emd_3418.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomogram average of the 80S ribosome obtained with the VPP
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.62 Å/pix.
x 160 pix.
= 419.2 Å		2.62 Å/pix.
x 160 pix.
= 419.2 Å		2.62 Å/pix.
x 160 pix.
= 419.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.62 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.24 / Movie #1: 3.24
Minimum - Maximum-6.65906477 - 12.266580579999999
Average (Standard dev.)-0.00000001 (±0.99999988)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 419.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.622.622.62
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z419.200419.200419.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-6.65912.267-0.000

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Supplemental data

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Sample components

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Entire : 80S ribosome

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

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Supramolecule #1000: 80S ribosome

SupramoleculeName: 80S ribosome / type: sample / ID: 1000 / Number unique components: 1

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Supramolecule #1: 80S ribosome

SupramoleculeName: 80S ribosome / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: ALL
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: Rabbit / Cell: Reticulocyte / Location in cell: Cytoplasm

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.6 / Details: 20mM Hepes, 50mM KCl; 2mM MgCl2
GridDetails: Quantifoil R 2/1
VitrificationCryogen name: ETHANE-PROPANE MIXTURE / Instrument: FEI VITROBOT MARK IV
Method: Blotting time of 3 seconds and a blot force of 0 before plunging

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Electron microscopy

MicroscopeFEI TITAN
Specialist opticsEnergy filter - Name: GIF Quantum / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
DateMay 6, 2015
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number real images: 80 / Average electron dose: 30 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 0.0 µm / Nominal defocus min: 0.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt series - Axis1 - Min angle: -20 ° / Tilt series - Axis1 - Max angle: 20 °

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 9.6 Å / Resolution method: OTHER / Software - Name: TOM, AV3, PyTom / Number subtomograms used: 1400
CTF correctionDetails: each tilt image

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