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- EMDB-10882: 70S ribosome prepared by blotting -

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Basic information

Entry
Database: EMDB / ID: EMD-10882
Title70S ribosome prepared by blotting
Map dataunsharpened final map
Sample
  • Complex: 70S ribosome from E. coliRibosome
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.1 Å
AuthorsKlebl DP / Gravett MSC / Darrow M / Thompson RF / Muench SP
Funding support United Kingdom, 3 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/P020208/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/P026397/1 United Kingdom
Wellcome Trust108466/Z/15/Z United Kingdom
CitationJournal: Structure / Year: 2020
Title: Need for Speed: Examining Protein Behavior during CryoEM Grid Preparation at Different Timescales.
Authors: David P Klebl / Molly S C Gravett / Dimitrios Kontziampasis / David J Wright / Robin S Bon / Diana C F Monteiro / Martin Trebbin / Frank Sobott / Howard D White / Michele C Darrow / Rebecca ...Authors: David P Klebl / Molly S C Gravett / Dimitrios Kontziampasis / David J Wright / Robin S Bon / Diana C F Monteiro / Martin Trebbin / Frank Sobott / Howard D White / Michele C Darrow / Rebecca F Thompson / Stephen P Muench /
Abstract: A host of new technologies are under development to improve the quality and reproducibility of cryoelectron microscopy (cryoEM) grid preparation. Here we have systematically investigated the ...A host of new technologies are under development to improve the quality and reproducibility of cryoelectron microscopy (cryoEM) grid preparation. Here we have systematically investigated the preparation of three macromolecular complexes using three different vitrification devices (Vitrobot, chameleon, and a time-resolved cryoEM device) on various timescales, including grids made within 6 ms (the fastest reported to date), to interrogate particle behavior at the air-water interface for different timepoints. Results demonstrate that different macromolecular complexes can respond to the thin-film environment formed during cryoEM sample preparation in highly variable ways, shedding light on why cryoEM sample preparation can be difficult to optimize. We demonstrate that reducing time between sample application and vitrification is just one tool to improve cryoEM grid quality, but that it is unlikely to be a generic "silver bullet" for improving the quality of every cryoEM sample preparation.
History
DepositionApr 17, 2020-
Header (metadata) releaseAug 5, 2020-
Map releaseAug 5, 2020-
UpdateMay 26, 2021-
Current statusMay 26, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.028
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.028
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10882.png

Downloads & links

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Map

FileDownload / File: emd_10882.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationunsharpened final map
Voxel sizeX=Y=Z: 1.065 Å
Density
Contour LevelBy AUTHOR: 0.028 / Movie #1: 0.028
Minimum - Maximum-0.019051591 - 0.07340058
Average (Standard dev.)-0.00013849506 (±0.010496371)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 319.50003 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0651.0651.065
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z319.500319.500319.500
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0190.073-0.000

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Supplemental data

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Mask #1

Fileemd_10882_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_10882_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_10882_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : 70S ribosome from E. coli

EntireName: 70S ribosome from E. coliRibosome
Components
  • Complex: 70S ribosome from E. coliRibosome

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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)

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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

Concentration1.1 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV / Details: blot time 6 s blot force 6.
Detailsthe sample was a mixture of 30S, 50S and 70S ribosomes, purchased from New England Biolabs (P0763S)

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Average exposure time: 1.5 sec. / Average electron dose: 78.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Details: The final angular assignment was done in a bigger, combined dataset which was then split into the original datasets to generate this reconstruction
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 5.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 22057
FSC plot (resolution estimation)

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