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- EMDB-23357: Structure of 80S ribosome from EMPIAR-10064 at 5.6 Angstrom Resolution -

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Basic information

Entry
Database: EMDB / ID: EMD-23357
TitleStructure of 80S ribosome from EMPIAR-10064 at 5.6 Angstrom Resolution
Map dataStructure of 80S ribosome from EMPIAR-10064 at 5.6 Angstrom Resolution
Sample
  • Complex: 80S ribsome from Oryctolagus cuniculus
Biological speciesOryctolagus cuniculus (rabbit)
Methodsubtomogram averaging / cryo EM / Resolution: 5.6 Å
AuthorsBouvette J / Liu HF / Du X / Zhou Y / Sikkema AP / Mello JFR / Klemm B / Huang R / Schaaper RM / Borgnia MJ / Bartesaghi A
CitationJournal: Nat Commun / Year: 2021
Title: Beam image-shift accelerated data acquisition for near-atomic resolution single-particle cryo-electron tomography.
Authors: Jonathan Bouvette / Hsuan-Fu Liu / Xiaochen Du / Ye Zhou / Andrew P Sikkema / Juliana da Fonseca Rezende E Mello / Bradley P Klemm / Rick Huang / Roel M Schaaper / Mario J Borgnia / Alberto Bartesaghi /
Abstract: Tomographic reconstruction of cryopreserved specimens imaged in an electron microscope followed by extraction and averaging of sub-volumes has been successfully used to derive atomic models of ...Tomographic reconstruction of cryopreserved specimens imaged in an electron microscope followed by extraction and averaging of sub-volumes has been successfully used to derive atomic models of macromolecules in their biological environment. Eliminating biochemical isolation steps required by other techniques, this method opens up the cell to in-situ structural studies. However, the need to compensate for errors in targeting introduced during mechanical navigation of the specimen significantly slows down tomographic data collection thus limiting its practical value. Here, we introduce protocols for tilt-series acquisition and processing that accelerate data collection speed by up to an order of magnitude and improve map resolution compared to existing approaches. We achieve this by using beam-image shift to multiply the number of areas imaged at each stage position, by integrating geometrical constraints during imaging to achieve high precision targeting, and by performing per-tilt astigmatic CTF estimation and data-driven exposure weighting to improve final map resolution. We validated our beam image-shift electron cryo-tomography (BISECT) approach by determining the structure of a low molecular weight target (~300 kDa) at 3.6 Å resolution where density for individual side chains is clearly resolved.
History
DepositionJan 26, 2021-
Header (metadata) releaseMar 3, 2021-
Map releaseMar 3, 2021-
UpdateApr 14, 2021-
Current statusApr 14, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.027
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.027
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23357.png

Downloads & links

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Map

FileDownload / File: emd_23357.map.gz / Format: CCP4 / Size: 5.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of 80S ribosome from EMPIAR-10064 at 5.6 Angstrom Resolution
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
2.62 Å/pix.
x 114 pix.
= 298.68 Å		2.62 Å/pix.
x 106 pix.
= 277.72 Å		2.62 Å/pix.
x 129 pix.
= 337.98 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 2.62 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.027 / Movie #1: 0.027
Minimum - Maximum-0.1837952 - 0.20162033
Average (Standard dev.)-2.9403872e-13 (±0.018231615)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin825064
Dimensions106129114
Spacing114106129
CellA: 298.68 Å / B: 277.72 Å / C: 337.97998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.622.622.62
M x/y/z114106129
origin x/y/z0.0000.0000.000
length x/y/z298.680277.720337.980
α/β/γ90.00090.00090.000
start NX/NY/NZ648250
NX/NY/NZ114106129
MAP C/R/S321
start NC/NR/NS508264
NC/NR/NS129106114
D min/max/mean-0.1840.202-0.000

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Supplemental data

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Mask #1

Fileemd_23357_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Structure of 80S ribosome from EMPIAR-10064 at 5.6...

Fileemd_23357_additional_1.map
AnnotationStructure of 80S ribosome from EMPIAR-10064 at 5.6 Angstrom Resolution
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Structure of 80S ribosome from EMPIAR-10064 at 5.6...

Fileemd_23357_half_map_1.map
AnnotationStructure of 80S ribosome from EMPIAR-10064 at 5.6 Angstrom Resolution
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Structure of 80S ribosome from EMPIAR-10064 at 5.6...

Fileemd_23357_half_map_2.map
AnnotationStructure of 80S ribosome from EMPIAR-10064 at 5.6 Angstrom Resolution
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : 80S ribsome from Oryctolagus cuniculus

EntireName: 80S ribsome from Oryctolagus cuniculus
Components
  • Complex: 80S ribsome from Oryctolagus cuniculus

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Supramolecule #1: 80S ribsome from Oryctolagus cuniculus

SupramoleculeName: 80S ribsome from Oryctolagus cuniculus / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (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
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 5.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number subtomograms used: 4286
ExtractionNumber tomograms: 4 / Number images used: 4286 / Method: Manually picked
Final 3D classificationNumber classes: 1
Final angle assignmentType: PROJECTION MATCHING
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT

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