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- EMDB-4487: Structure of E. coli ribosome deposited by spraying -

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Basic information

Entry
Database: EMDB / ID: EMD-4487
TitleStructure of E. coli ribosome deposited by spraying
Map dataLocalRes filtered map
Sample
  • Complex: ribosome from E. coli
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsKontziampasis D / Klebl DP / Iadanza MG / Scarff CA / Kopf F / Sobott F / Monteiro DCF / Trebbin M / Muench SP / White HD
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research CouncilBB/P026397 United Kingdom
CitationJournal: IUCrJ / Year: 2019
Title: A cryo-EM grid preparation device for time-resolved structural studies.
Authors: Dimitrios Kontziampasis / David P Klebl / Matthew G Iadanza / Charlotte A Scarff / Florian Kopf / Frank Sobott / Diana C F Monteiro / Martin Trebbin / Stephen P Muench / Howard D White /
Abstract: Structural biology generally provides static snapshots of protein conformations that can provide information on the functional mechanisms of biological systems. Time-resolved structural biology ...Structural biology generally provides static snapshots of protein conformations that can provide information on the functional mechanisms of biological systems. Time-resolved structural biology provides a means to visualize, at near-atomic resolution, the dynamic conformational changes that macromolecules undergo as they function. X-ray free-electron-laser technology has provided a powerful tool to study enzyme mechanisms at atomic resolution, typically in the femtosecond to picosecond timeframe. Complementary to this, recent advances in the resolution obtainable by electron microscopy and the broad range of samples that can be studied make it ideally suited to time-resolved approaches in the microsecond to millisecond timeframe to study large loop and domain motions in biomolecules. Here we describe a cryo-EM grid preparation device that permits rapid mixing, voltage-assisted spraying and vitrification of samples. It is shown that the device produces grids of sufficient ice quality to enable data collection from single grids that results in a sub-4 Å reconstruction. Rapid mixing can be achieved by blot-and-spray or mix-and-spray approaches with a delay of ∼10 ms, providing greater temporal resolution than previously reported mix-and-spray approaches.
History
DepositionDec 19, 2018-
Header (metadata) releaseOct 2, 2019-
Map releaseOct 2, 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: 0.06
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4487.png

Downloads & links

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Map

FileDownload / File: emd_4487.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocalRes filtered 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: 0.06 / Movie #1: 0.06
Minimum - Maximum-0.18893075 - 0.35352272
Average (Standard dev.)0.0017891114 (±0.019610811)
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
start NX/NY/NZ000
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.1890.3540.002

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Supplemental data

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Additional map: Sharpened map

Fileemd_4487_additional_1.map
AnnotationSharpened map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map

Fileemd_4487_additional_2.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap 1

Fileemd_4487_half_map_1.map
Annotationhalfmap 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap 2

Fileemd_4487_half_map_2.map
Annotationhalfmap 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : ribosome from E. coli

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

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Supramolecule #1: ribosome from E. coli

SupramoleculeName: 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

Concentration1.9 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
100.0 mMCH3CO2Kpotassium acetate
50.0 mMC8H18N2O4Shepes
8.0 mMMg(CH3COO)2magnesium acetate
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 293 K / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Average electron dose: 66.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / 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: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3) / Number images used: 34010
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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