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- EMDB-10875: 50S ribosome subunit deposited by spraying (13 ms delay) -

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Basic information

Entry
Database: EMDB / ID: EMD-10875
Title50S ribosome subunit deposited by spraying (13 ms delay)
Map dataunsharpened final map
Sample
  • Complex: 50S ribosome from E. coli
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: 0.03
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.03
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10875.png

Downloads & links

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Map

FileDownload / File: emd_10875.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationunsharpened final map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 300 pix.
= 319.5 Å		1.07 Å/pix.
x 300 pix.
= 319.5 Å		1.07 Å/pix.
x 300 pix.
= 319.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.065 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.019040996 - 0.0718297
Average (Standard dev.)-2.810493e-05 (±0.008472842)
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-0.0190.072-0.000

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Supplemental data

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Mask #1

Fileemd_10875_msk_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_10875_half_map_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_10875_half_map_2.map
Projections & Slices
AxesZYX

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Density Histograms

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Histogram (log scale)

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Sample components

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Entire : 50S ribosome from E. coli

EntireName: 50S ribosome from E. coli
Components
  • Complex: 50S ribosome from E. coli

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Supramolecule #1: 50S ribosome from E. coli

SupramoleculeName: 50S 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

Concentration3.3 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 293 K / Instrument: HOMEMADE PLUNGER
Details: the sample was sprayed using a microfludic nozzle, resulting in a delay of 13ms between spraying and freezing.
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
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Average exposure time: 1.5 sec. / Average electron dose: 77.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

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: 15871
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
FSC plot (resolution estimation)

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