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- EMDB-5905: Cryo-EM structure of low salt treated immature 30S ribosomal subu... -

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Entry
Database: EMDB / ID: EMD-5905
TitleCryo-EM structure of low salt treated immature 30S ribosomal subunit from rsga and rbfa deleted E.coli strain
Map data
SampleCryo-EM structure of low salt treated immature 30S ribosomal subunit from rsga and rbfa deleted E.coli strain:
ribosome-prokaryote
Keywords30S subunit assembly / RsgA / RbfA / 17S rRNA processing
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 14.4 Å
AuthorsYang Z / Guo Q / Goto S / Chen Y / Li N / Yan K / Zhang Y / Muto A / Deng H / Himeno H ...Yang Z / Guo Q / Goto S / Chen Y / Li N / Yan K / Zhang Y / Muto A / Deng H / Himeno H / Lei J / Gao N
CitationJournal: Protein Cell / Year: 2014
Title: Structural insights into the assembly of the 30S ribosomal subunit in vivo: functional role of S5 and location of the 17S rRNA precursor sequence.
Authors: Zhixiu Yang / Qiang Guo / Simon Goto / Yuling Chen / Ningning Li / Kaige Yan / Yixiao Zhang / Akira Muto / Haiteng Deng / Hyouta Himeno / Jianlin Lei / Ning Gao /
Abstract: The in vivo assembly of ribosomal subunits is a highly complex process, with a tight coordination between protein assembly and rRNA maturation events, such as folding and processing of rRNA ...The in vivo assembly of ribosomal subunits is a highly complex process, with a tight coordination between protein assembly and rRNA maturation events, such as folding and processing of rRNA precursors, as well as modifications of selected bases. In the cell, a large number of factors are required to ensure the efficiency and fidelity of subunit production. Here we characterize the immature 30S subunits accumulated in a factor-null Escherichia coli strain (∆rsgA∆rbfA). The immature 30S subunits isolated with varying salt concentrations in the buffer system show interesting differences on both protein composition and structure. Specifically, intermediates derived under the two contrasting salt conditions (high and low) likely reflect two distinctive assembly stages, the relatively early and late stages of the 3' domain assembly, respectively. Detailed structural analysis demonstrates a mechanistic coupling between the maturation of the 5' end of the 17S rRNA and the assembly of the 30S head domain, and attributes a unique role of S5 in coordinating these two events. Furthermore, our structural results likely reveal the location of the unprocessed terminal sequences of the 17S rRNA, and suggest that the maturation events of the 17S rRNA could be employed as quality control mechanisms on subunit production and protein translation.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionFeb 12, 2014-
Header (metadata) releaseMar 5, 2014-
Map releaseApr 9, 2014-
UpdateJun 11, 2014-
Current statusJun 11, 2014Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.75
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 2.75
  • Imaged by UCSF Chimera
  • Download
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Structure viewerEM map:
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Map

FileDownload / File: emd_5905.map.gz / Format: CCP4 / Size: 7.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3 Å/pix.
x 124 pix.
= 372. Å
3 Å/pix.
x 124 pix.
= 372. Å
3 Å/pix.
x 124 pix.
= 372. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3 Å
Density
Contour LevelBy AUTHOR: 2.75 / Movie #1: 2.75
Minimum - Maximum-2.99743819 - 13.06661892
Average (Standard dev.)0.0 (±0.99999976)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-61-61-61
Dimensions124124124
Spacing124124124
CellA=B=C: 372.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z333
M x/y/z124124124
origin x/y/z0.0000.0000.000
length x/y/z372.000372.000372.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-95-75153
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS-61-61-61
NC/NR/NS124124124
D min/max/mean-2.99713.067-0.000

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Supplemental data

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Sample components

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Entire Cryo-EM structure of low salt treated immature 30S ribosomal subu...

EntireName: Cryo-EM structure of low salt treated immature 30S ribosomal subunit from rsga and rbfa deleted E.coli strain
Number of components: 1
MassTheoretical: 800 kDa / Experimental: 800 kDa

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Component #1: ribosome-prokaryote, low salt treated immature 30S ribosomal subu...

Ribosome-prokaryoteName: low salt treated immature 30S ribosomal subunit from rsga and rbfa deleted E.coli strain
a.k.a: immature 30S / Prokaryote: SSU 30S / Recombinant expression: No
MassTheoretical: 800 kDa / Experimental: 800 kDa
SourceSpecies: Escherichia coli (E. coli) / Strain: A19

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: negative staining, cryo EM
Sample solutionBuffer solution: 20mM Tris-HCl, 150mM NH4Cl,10mM Mg(OAc)2 / pH: 7.5
Support filmQuantifoil 2/4 grids were coated with carbon and glow discharged in a Harrick Plasma Cleaner for 30 seconds
Staininggrids were prepared with an FEI Vitrobot Mark IV
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Method: blot for 1 second before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Jul 14, 2012
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 20 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 59000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000 - 8500 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: FEI EAGLE (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 4365

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 42521
Details: this is a classification volume (NO.3 of five groups) using RELION
3D reconstructionAlgorithm: reference projection / Software: SPIDER / CTF correction: weiner filter / Resolution: 14.4 Å / Resolution method: FSC 0.5, semi-independent

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