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- EMDB-10211: Subtomogram average of E. coli 70S ribosome from FISE tilt series -

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Basic information

Entry
Database: EMDB / ID: EMD-10211
TitleSubtomogram average of E. coli 70S ribosome from FISE tilt series
Map data
Sample
  • Complex: 70S ribosome
Biological speciesEscherichia coli DH5[alpha] (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 9.0 Å
AuthorsEisenstein F / Danev R / Pilhofer M
Citation
Journal: J Struct Biol / Year: 2019
Title: Improved applicability and robustness of fast cryo-electron tomography data acquisition.
Authors: Fabian Eisenstein / Radostin Danev / Martin Pilhofer /
Abstract: The power of cryo-electron tomography (cryoET) lies in its capability to characterize macromolecules in their cellular context. Structure determination by cryoET, however, is time-consuming compared ...The power of cryo-electron tomography (cryoET) lies in its capability to characterize macromolecules in their cellular context. Structure determination by cryoET, however, is time-consuming compared to single particle approaches. A recent study reported significant acceleration of data acquisition by a fast-incremental single-exposure (FISE) tilt series scheme. Here we improved the method and evaluated its efficiency and performance. We show that (1) FISE combined with the latest generation of direct electron detectors speeds up collection considerably, (2) previous generation (pre-2017) double-tilt axis Titan Krios holders are also suitable for FISE data acquisition, (3) x, y and z-specimen shifts can be compensated for, and (4) FISE tilt series data can generate averages of sub-nanometer resolution. These advances will allow for a widespread adoption of cryoET for high-throughput in situ studies and high-resolution structure determination across different biological research disciplines.
#2: Journal: J.Struct.Biol. / Year: 2019
Title: Improved applicability and robustness of fast cryo-electron tomography data acquisition
Authors: Eisenstein F / Danev R / Pilhofer M
History
DepositionAug 12, 2019-
Header (metadata) releaseAug 28, 2019-
Map releaseAug 28, 2019-
UpdateNov 6, 2019-
Current statusNov 6, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10211.png

Downloads & links

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Map

FileDownload / File: emd_10211.map.gz / Format: CCP4 / Size: 48.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 2.1 Å
Density
Contour LevelBy AUTHOR: 0.43 / Movie #1: 2
Minimum - Maximum-6.6730123 - 15.664622
Average (Standard dev.)0.06573385 (±0.66045946)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions234234234
Spacing234234234
CellA=B=C: 491.39996 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.12.12.1
M x/y/z234234234
origin x/y/z0.0000.0000.000
length x/y/z491.400491.400491.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS234234234
D min/max/mean-6.67315.6650.066

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Supplemental data

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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 DH5[alpha] (bacteria)

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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 R2/2 / Material: COPPER / Mesh: 200
VitrificationCryogen name: ETHANE / Chamber humidity: 100 %

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Detailssingle-tilt axis holder
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average exposure time: 0.5 sec. / Average electron dose: 150.0 e/Å2 / Details: FISEtomo SerialEM script
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber classes used: 3 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 9.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: emClarity (ver. 1.3.9) / Number subtomograms used: 9923
ExtractionNumber tomograms: 12 / Number images used: 11251 / Reference model: EMD-6311 / Method: template matching / Software - Name: emClarity (ver. 1.3.9) / Details: emClarity
CTF correctionSoftware - Name: emClarity (ver. 1.3.9)
Final 3D classificationNumber classes: 4 / Software - Name: emClarity (ver. 1.3.9)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: emClarity (ver. 1.3.9)

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