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- EMDB-10265: Structure of E. coli 70S ribosome determined at 100 keV -

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Basic information

Entry
Database: EMDB / ID: EMD-10265
TitleStructure of E. coli 70S ribosome determined at 100 keV
Map dataMasked and sharpened (B = -150 A^2) map
Sample
  • Complex: 70S Ribosome from E. coli
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.0 Å
AuthorsNaydenova K / McMullan G / Peet MJ / Lee Y / Edwards PC / Chen S / Leahy E / Henderson R / Russo CJ
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC_UP_120117 United Kingdom
Medical Research Council (United Kingdom)MC_U105184322 United Kingdom
CitationJournal: IUCrJ / Year: 2019
Title: CryoEM at 100 keV: a demonstration and prospects.
Authors: K Naydenova / G McMullan / M J Peet / Y Lee / P C Edwards / S Chen / E Leahy / S Scotcher / R Henderson / C J Russo /
Abstract: 100 kV is investigated as the operating voltage for single-particle electron cryomicroscopy (cryoEM). Reducing the electron energy from the current standard of 300 or 200 keV offers both cost ...100 kV is investigated as the operating voltage for single-particle electron cryomicroscopy (cryoEM). Reducing the electron energy from the current standard of 300 or 200 keV offers both cost savings and potentially improved imaging. The latter follows from recent measurements of radiation damage to biological specimens by high-energy electrons, which show that at lower energies there is an increased amount of information available per unit damage. For frozen hydrated specimens around 300 Å in thickness, the predicted optimal electron energy for imaging is 100 keV. Currently available electron cryomicroscopes in the 100-120 keV range are not optimized for cryoEM as they lack both the spatially coherent illumination needed for the high defocus used in cryoEM and imaging detectors optimized for 100 keV electrons. To demonstrate the potential of imaging at 100 kV, the voltage of a standard, commercial 200 kV field-emission gun (FEG) microscope was reduced to 100 kV and a side-entry cryoholder was used. As high-efficiency, large-area cameras are not currently available for 100 keV electrons, a commercial hybrid pixel camera designed for X-ray detection was attached to the camera chamber and was used for low-dose data collection. Using this configuration, five single-particle specimens were imaged: hepatitis B virus capsid, bacterial 70S ribosome, catalase, DNA protection during starvation protein and haemoglobin, ranging in size from 4.5 MDa to 64 kDa with corresponding diameters from 320 to 72 Å. These five data sets were used to reconstruct 3D structures with resolutions between 8.4 and 3.4 Å. Based on this work, the practical advantages and current technological limitations to single-particle cryoEM at 100 keV are considered. These results are also discussed in the context of future microscope development towards the goal of rapid, simple and widely available structure determination of any purified biological specimen.
History
DepositionAug 28, 2019-
Header (metadata) releaseNov 20, 2019-
Map releaseNov 20, 2019-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.3
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10265.png

Downloads & links

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Map

FileDownload / File: emd_10265.map.gz / Format: CCP4 / Size: 10.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMasked and sharpened (B = -150 A^2) map
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.74 Å/pix.
x 140 pix.
= 383.6 Å		2.74 Å/pix.
x 140 pix.
= 383.6 Å		2.74 Å/pix.
x 140 pix.
= 383.6 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 2.74 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3 / Movie #1: 0.3
Minimum - Maximum-0.17475282 - 0.76398116
Average (Standard dev.)0.016547212 (±0.08402181)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions140140140
Spacing140140140
CellA=B=C: 383.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.742.742.74
M x/y/z140140140
origin x/y/z0.0000.0000.000
length x/y/z383.600383.600383.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS140140140
D min/max/mean-0.1750.7640.017

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Supplemental data

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Mask #1

Fileemd_10265_msk_1.map
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Additional map: Unfiltered map

Fileemd_10265_additional.map
AnnotationUnfiltered map
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Additional map: Unfiltered map

Fileemd_10265_additional_1.map
AnnotationUnfiltered map
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Half map: Half map 2

Fileemd_10265_half_map_1.map
AnnotationHalf map 2
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Half map: Half map 1

Fileemd_10265_half_map_2.map
AnnotationHalf map 1
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Sample components

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Entire : 70S Ribosome from E. coli

EntireName: 70S Ribosome from E. coli
Components
  • Complex: 70S Ribosome from E. coli

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Supramolecule #1: 70S Ribosome from E. coli

SupramoleculeName: 70S Ribosome from E. coli / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MRE600
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: MRE600

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.7
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TECNAI F20
Image recordingFilm or detector model: OTHER / Average electron dose: 24.64 e/Å2
Electron beamAcceleration voltage: 100 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 3644
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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