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- EMDB-1413: Structural aspects of RbfA action during small ribosomal subunit ... -

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Basic information

Entry
Database: EMDB / ID: EMD-1413
TitleStructural aspects of RbfA action during small ribosomal subunit assembly.
Map dataCryo-EM map of Thermus thermophilus 30S subunit and RbfA complex.
Sample
  • Sample: Complex of ribosomal subunit 30S and RbfA
  • Complex: 30S small ribosomal subunit
  • Protein or peptide: Ribosome binding factor A
Function / homology
Function and homology information


maturation of SSU-rRNA / small ribosomal subunit / tRNA binding / rRNA binding / ribosome / structural constituent of ribosome / translation / ribonucleoprotein complex / response to antibiotic / cytosol / cytoplasm
Similarity search - Function
Ribosome-binding factor A / Ribosome-binding factor A domain superfamily / Ribosome-binding factor A / Ribosomal protein S13, bacterial-type / Ribosomal protein S9, bacterial/plastid / Ribosomal protein S12, bacterial-type / Ribosomal protein S13, conserved site / Ribosomal protein S13 signature. / Ribosomal protein S13 / 30s ribosomal protein S13, C-terminal ...Ribosome-binding factor A / Ribosome-binding factor A domain superfamily / Ribosome-binding factor A / Ribosomal protein S13, bacterial-type / Ribosomal protein S9, bacterial/plastid / Ribosomal protein S12, bacterial-type / Ribosomal protein S13, conserved site / Ribosomal protein S13 signature. / Ribosomal protein S13 / 30s ribosomal protein S13, C-terminal / Ribosomal protein S13/S18 / Ribosomal protein S13 family profile. / K homology domain-like, alpha/beta / Ribosomal protein S9, conserved site / Ribosomal protein S9 signature. / Ribosomal protein S13-like, H2TH / Ribosomal protein S12 signature. / Ribosomal protein S9 / Ribosomal protein S9/S16 / Ribosomal protein S12/S23 / Ribosomal protein S12/S23 / Ribosomal protein S5 domain 2-type fold, subgroup / Ribosomal protein S5 domain 2-type fold / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
Small ribosomal subunit protein uS12 / Small ribosomal subunit protein uS12 / Small ribosomal subunit protein uS13 / Small ribosomal subunit protein uS9 / Ribosome-binding factor A
Similarity search - Component
Biological speciesThermus thermophilus HB8 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 12.5 Å
AuthorsDatta PP / Wilson DN / Kawazoe M / Swami NK / Kaminishi T / Sharma MR / Booth TM / Takemoto C / Fucini P / Yokoyama S / Agrawal RK
CitationJournal: Mol Cell / Year: 2007
Title: Structural aspects of RbfA action during small ribosomal subunit assembly.
Authors: Partha P Datta / Daniel N Wilson / Masahito Kawazoe / Neil K Swami / Tatsuya Kaminishi / Manjuli R Sharma / Timothy M Booth / Chie Takemoto / Paola Fucini / Shigeyuki Yokoyama / Rajendra K Agrawal /
Abstract: Ribosome binding factor A (RbfA) is a bacterial cold shock response protein, required for an efficient processing of the 5' end of the 16S ribosomal RNA (rRNA) during assembly of the small (30S) ...Ribosome binding factor A (RbfA) is a bacterial cold shock response protein, required for an efficient processing of the 5' end of the 16S ribosomal RNA (rRNA) during assembly of the small (30S) ribosomal subunit. Here we present a crystal structure of Thermus thermophilus (Tth) RbfA and a three-dimensional cryo-electron microscopic (EM) map of the Tth 30S*RbfA complex. RbfA binds to the 30S subunit in a position overlapping the binding sites of the A and P site tRNAs, and RbfA's functionally important C terminus extends toward the 5' end of the 16S rRNA. In the presence of RbfA, a portion of the 16S rRNA encompassing helix 44, which is known to be directly involved in mRNA decoding and tRNA binding, is displaced. These results shed light on the role played by RbfA during maturation of the 30S subunit, and also indicate how RbfA provides cells with a translational advantage under conditions of cold shock.
History
DepositionAug 20, 2007-
Header (metadata) releaseAug 21, 2007-
Map releaseNov 16, 2007-
UpdateOct 17, 2012-
Current statusOct 17, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 35
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 35
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-2r1c, PDB-2r1g
  • Surface level: 35
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-2r1c
  • Imaged by Jmol
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-2r1g
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_1413.map.gz / Format: CCP4 / Size: 8.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of Thermus thermophilus 30S subunit and RbfA complex.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.76 Å/pix.
x 130 pix.
= 358.8 Å
2.76 Å/pix.
x 130 pix.
= 358.8 Å
2.76 Å/pix.
x 130 pix.
= 358.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.76 Å
Density
Contour Level1: 36.700000000000003 / Movie #1: 35
Minimum - Maximum-130.322000000000003 - 407.386000000000024
Average (Standard dev.)2.66509 (±22.7014)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-65-65-65
Dimensions130130130
Spacing130130130
CellA=B=C: 358.8 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.762.762.76
M x/y/z130130130
origin x/y/z0.0000.0000.000
length x/y/z358.800358.800358.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-90-90-90
NX/NY/NZ181181181
MAP C/R/S123
start NC/NR/NS-65-65-65
NC/NR/NS130130130
D min/max/mean-130.322407.3862.665

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Supplemental data

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Sample components

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Entire : Complex of ribosomal subunit 30S and RbfA

EntireName: Complex of ribosomal subunit 30S and RbfA
Components
  • Sample: Complex of ribosomal subunit 30S and RbfA
  • Complex: 30S small ribosomal subunit
  • Protein or peptide: Ribosome binding factor A

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Supramolecule #1000: Complex of ribosomal subunit 30S and RbfA

SupramoleculeName: Complex of ribosomal subunit 30S and RbfA / type: sample / ID: 1000 / Number unique components: 2
Molecular weightTheoretical: 910.9 KDa

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Supramolecule #1: 30S small ribosomal subunit

SupramoleculeName: 30S small ribosomal subunit / type: complex / ID: 1 / Name.synonym: 30S / Details: Thermus thermophilus / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: SSU 30S
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightExperimental: 900 KDa

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Macromolecule #1: Ribosome binding factor A

MacromoleculeName: Ribosome binding factor A / type: protein_or_peptide / ID: 1 / Name.synonym: RbfA / Recombinant expression: Yes
Source (natural)Organism: Thermus thermophilus HB8 (bacteria) / Strain: HB8
Molecular weightExperimental: 10.9 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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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: 7.8
Details: 20mM Hepes-KOH (pH7.8), 10mM Mg(OAc)2, 200 mM NH4Cl, 65 mM KCL.
GridDetails: Quantifoil 300 mesh Copper grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: Wadworth Center own cryo-plunger fabricated on-site
Method: 5ul of specimen was applied to the grid, then blotted using Whatman number 1 filter paper for 2 to 4 seconds, then plunged.

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureAverage: 93 K
Alignment procedureLegacy - Astigmatism: Objective lends astigmatism corrected at 210x magnification
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 14 µm / Number real images: 131 / Average electron dose: 20 e/Å2 / Bits/pixel: 12
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50760 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.7 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Oxford cryo-transfer holder / Specimen holder model: OTHER
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 12.5 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Number images used: 61207

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Atomic model buiding 1

SoftwareName: O
DetailsProtocol: Rigid Body. The domains were fitted independently using the program O
RefinementProtocol: RIGID BODY FIT
Output model

PDB-2r1c:
Coordinates of the thermus thermophilus ribosome binding factor A (RbfA) homology model as fitted into the CRYO-EM map of a 30S-RBFA complex

PDB-2r1g:
Coordinates of the thermus thermophilus 30S components neighboring RbfA as obtained by fitting into the CRYO-EM map of A 30S-RBFA complex

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