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- EMDB-2970: Cryo-EM structure of E. coli 70S ribosome bound to additional non... -

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Basic information

Entry
Database: EMDB / ID: EMD-2970
TitleCryo-EM structure of E. coli 70S ribosome bound to additional non-ribosomal proteins.
Map dataCryo-EM structure of E. coli 70S ribosome bound to additional non-ribosomal proteins. MapI
Sample
  • Sample: E. coli 70S ribosome
  • Complex: Escherichia coli 70S ribosome
Keywordsribosome / ribosome assembly / moonlighting proteins.
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 12.9 Å
AuthorsShasmal M / Dey S / Shaikh TR / Bhakta S / Sengupta J
CitationJournal: Sci Rep / Year: 2016
Title: E. coli metabolic protein aldehyde-alcohol dehydrogenase-E binds to the ribosome: a unique moonlighting action revealed.
Authors: Manidip Shasmal / Sandip Dey / Tanvir R Shaikh / Sayan Bhakta / Jayati Sengupta /
Abstract: It is becoming increasingly evident that a high degree of regulation is involved in the protein synthesis machinery entailing more interacting regulatory factors. A multitude of proteins have been ...It is becoming increasingly evident that a high degree of regulation is involved in the protein synthesis machinery entailing more interacting regulatory factors. A multitude of proteins have been identified recently which show regulatory function upon binding to the ribosome. Here, we identify tight association of a metabolic protein aldehyde-alcohol dehydrogenase E (AdhE) with the E. coli 70S ribosome isolated from cell extract under low salt wash conditions. Cryo-EM reconstruction of the ribosome sample allows us to localize its position on the head of the small subunit, near the mRNA entrance. Our study demonstrates substantial RNA unwinding activity of AdhE which can account for the ability of ribosome to translate through downstream of at least certain mRNA helices. Thus far, in E. coli, no ribosome-associated factor has been identified that shows downstream mRNA helicase activity. Additionally, the cryo-EM map reveals interaction of another extracellular protein, outer membrane protein C (OmpC), with the ribosome at the peripheral solvent side of the 50S subunit. Our result also provides important insight into plausible functional role of OmpC upon ribosome binding. Visualization of the ribosome purified directly from the cell lysate unveils for the first time interactions of additional regulatory proteins with the ribosome.
History
DepositionMar 31, 2015-
Header (metadata) releaseMay 6, 2015-
Map releaseFeb 10, 2016-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 14
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 14
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Image_MapI_E...

Downloads & links

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Map

FileDownload / File: emd_2970.map.gz / Format: CCP4 / Size: 45.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of E. coli 70S ribosome bound to additional non-ribosomal proteins. MapI
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.69 Å/pix.
x 230 pix.
= 388.7 Å		1.69 Å/pix.
x 230 pix.
= 388.7 Å		1.69 Å/pix.
x 230 pix.
= 388.7 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.69 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 14.0 / Movie #1: 14
Minimum - Maximum-129.547348019999987 - 360.919616700000006
Average (Standard dev.)3.2442615 (±36.168273929999998)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions230230230
Spacing230230230
CellA=B=C: 388.7 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.691.691.69
M x/y/z230230230
origin x/y/z0.0000.0000.000
length x/y/z388.700388.700388.700
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS230230230
D min/max/mean-129.547360.9203.244

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Supplemental data

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Sample components

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Entire : E. coli 70S ribosome

EntireName: E. coli 70S ribosome
Components
  • Sample: E. coli 70S ribosome
  • Complex: Escherichia coli 70S ribosome

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Supramolecule #1000: E. coli 70S ribosome

SupramoleculeName: E. coli 70S ribosome / type: sample / ID: 1000 / Details: Monodisperse, single particle form / Number unique components: 1
Molecular weightTheoretical: 2.5 MDa

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Supramolecule #1: Escherichia coli 70S ribosome

SupramoleculeName: Escherichia coli 70S ribosome / type: complex / ID: 1 / Name.synonym: 70S ribosome / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: ALL
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MRE600
Molecular weightTheoretical: 2.5 MDa

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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: 7.5
Details: 20 mM Tris-HCl, pH 7.5, 10 mM MgOAc, 30 mM NH4Cl, 5 mM 2-mercaptoethanol
GridDetails: 300 mesh carbon coated lacey copper grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 90 K / Instrument: GATAN CRYOPLUNGE 3 / Method: Blot for 4.3 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureMin: 85 K / Max: 95 K
Alignment procedureLegacy - Astigmatism: Astigmatism was corrected at 90000 magnification.
DetailsLow dose imaging
DateMar 21, 2013
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Digitization - Sampling interval: 4 µm / Number real images: 271 / Average electron dose: 15 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 88466 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1.2 mm / Nominal defocus max: 4.6 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 89000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsParticles are selected first automatically by SPIDER and then manually by Jweb, grouped on the basis of defocus values and aligned by projection matching
CTF correctionDetails: CTF correction of 3D map
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 12.9 Å / Resolution method: OTHER / Software - Name: SPIDER / Number images used: 56165

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Atomic model buiding 1

Initial modelPDB ID:

2j1n


Chain - Chain ID: A
SoftwareName: PyMOL, MDFF
DetailsFirst manual rigid body fitting followed by flexible fitting by MDFF.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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