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- EMDB-23358: Structure of E. coli 70S ribosome from EMPIAR-10304 at 4.8 Angstr... -

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Basic information

Entry
Database: EMDB / ID: EMD-23358
TitleStructure of E. coli 70S ribosome from EMPIAR-10304 at 4.8 Angstrom Resolution
Map dataStructure of E. coli 70S ribosome from EMPIAR-10304 at 4.8 Angstrom Resolution
Sample
  • Complex: 70S ribosome
Biological speciesEscherichia coli (E. coli)
Methodsubtomogram averaging / cryo EM / Resolution: 4.8 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: 0.04
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.04
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23358.png

Downloads & links

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Map

FileDownload / File: emd_23358.map.gz / Format: CCP4 / Size: 9.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of E. coli 70S ribosome from EMPIAR-10304 at 4.8 Angstrom Resolution
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
2.1 Å/pix.
x 139 pix.
= 291.9 Å		2.1 Å/pix.
x 143 pix.
= 300.3 Å		2.1 Å/pix.
x 124 pix.
= 260.4 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: 2.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.054 / Movie #1: 0.04
Minimum - Maximum-0.33342642 - 0.3659016
Average (Standard dev.)9.830587e-13 (±0.025375808)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin536564
Dimensions143124139
Spacing139143124
CellA: 291.9 Å / B: 300.3 Å / C: 260.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.12.12.1
M x/y/z139143124
origin x/y/z0.0000.0000.000
length x/y/z291.900300.300260.400
α/β/γ90.00090.00090.000
start NX/NY/NZ645365
NX/NY/NZ139143124
MAP C/R/S321
start NC/NR/NS655364
NC/NR/NS124143139
D min/max/mean-0.3330.3660.000

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Supplemental data

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Mask #1

Fileemd_23358_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Structure of E. coli 70S ribosome from EMPIAR-10304...

Fileemd_23358_additional_1.map
AnnotationStructure of E. coli 70S ribosome from EMPIAR-10304 at 4.8 Angstrom Resolution
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Structure of E. coli 70S ribosome from EMPIAR-10304...

Fileemd_23358_half_map_1.map
AnnotationStructure of E. coli 70S ribosome from EMPIAR-10304 at 4.8 Angstrom Resolution
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Structure of E. coli 70S ribosome from EMPIAR-10304...

Fileemd_23358_half_map_2.map
AnnotationStructure of E. coli 70S ribosome from EMPIAR-10304 at 4.8 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 : 70S ribosome

EntireName: 70S ribosome
Components
  • Complex: 70S ribosome

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Supramolecule #1: 70S ribosome

SupramoleculeName: 70S ribosome / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli (E. coli)
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
GridModel: Quantifoil / Material: COPPER / Mesh: 200
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 4092 pixel / Digitization - Dimensions - Height: 5760 pixel / Average electron dose: 3.0 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: 4.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number subtomograms used: 12914
ExtractionNumber tomograms: 12 / Number images used: 12914
Final 3D classificationNumber classes: 1
Final angle assignmentType: PROJECTION MATCHING
FSC plot (resolution estimation)

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