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- EMDB-1009: A cryo-electron microscopic study of ribosome-bound termination f... -

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Basic information

Entry
Database: EMDB / ID: EMD-1009
TitleA cryo-electron microscopic study of ribosome-bound termination factor RF2.
Map dataThe RF2(GAQ) mutant cryo-density from the 3D-EM map of RF2 mutant bound to the E.coli 70s ribosome
Sample
  • Sample: RF2 (GAQ) mutant from E.coli
  • Protein or peptide: RF2 (GAQ) mutant
Function / homology
Function and homology information


translation release factor activity, codon specific / translational termination / large ribosomal subunit rRNA binding / small ribosomal subunit / cytosolic large ribosomal subunit / tRNA binding / rRNA binding / structural constituent of ribosome / translation / viral translational frameshifting / cytosol
Similarity search - Function
Peptide chain release factor 2 / Peptide chain release factor / PCRF domain / PCRF / Peptide chain release factor class I superfamily / Prokaryotic-type class I peptide chain release factors signature. / Peptide chain release factor class I / RF-1 domain / Ribosomal protein L11, bacterial-type / Ribosomal protein L11, conserved site ...Peptide chain release factor 2 / Peptide chain release factor / PCRF domain / PCRF / Peptide chain release factor class I superfamily / Prokaryotic-type class I peptide chain release factors signature. / Peptide chain release factor class I / RF-1 domain / Ribosomal protein L11, bacterial-type / Ribosomal protein L11, conserved site / Ribosomal protein L11 signature. / Ribosomal protein L11, N-terminal / Ribosomal protein L11, N-terminal domain / Ribosomal protein L11/L12 / Ribosomal protein L11, C-terminal / Ribosomal protein L11, C-terminal domain superfamily / Ribosomal protein L11/L12, N-terminal domain superfamily / Ribosomal protein L11/L12 / Ribosomal protein L11, RNA binding domain / Ribosomal protein S12, bacterial-type / Ribosomal protein S12 signature. / Ribosomal protein S12/S23 / Ribosomal protein S12/S23 / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
Peptide chain release factor RF2 / Large ribosomal subunit protein uL11 / Small ribosomal subunit protein uS12
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 10.9 Å
AuthorsRawat UB / Zavialov AV / Sengupta J / Valle M / Grassucci RA / Linde J / Vestergaard B / Ehrenberg M / Frank J
CitationJournal: Nature / Year: 2003
Title: A cryo-electron microscopic study of ribosome-bound termination factor RF2.
Authors: Urmila B S Rawat / Andrey V Zavialov / Jayati Sengupta / Mikel Valle / Robert A Grassucci / Jamie Linde / Bente Vestergaard / Måns Ehrenberg / Joachim Frank /
Abstract: Protein synthesis takes place on the ribosome, where genetic information carried by messenger RNA is translated into a sequence of amino acids. This process is terminated when a stop codon moves into ...Protein synthesis takes place on the ribosome, where genetic information carried by messenger RNA is translated into a sequence of amino acids. This process is terminated when a stop codon moves into the ribosomal decoding centre (DC) and is recognized by a class-1 release factor (RF). RFs have a conserved GGQ amino-acid motif, which is crucial for peptide release and is believed to interact directly with the peptidyl-transferase centre (PTC) of the 50S ribosomal subunit. Another conserved motif of RFs (SPF in RF2) has been proposed to interact directly with stop codons in the DC of the 30S subunit. The distance between the DC and PTC is approximately 73 A. However, in the X-ray structure of RF2, SPF and GGQ are only 23 A apart, indicating that they cannot be at DC and PTC simultaneously. Here we show that RF2 is in an open conformation when bound to the ribosome, allowing GGQ to reach the PTC while still allowing SPF-stop-codon interaction. The results indicate new interpretations of accuracy in termination, and have implications for how the presence of a stop codon in the DC is signalled to PTC.
History
DepositionSep 26, 2002-
Header (metadata) releaseSep 26, 2002-
Map releaseSep 26, 2003-
UpdateDec 4, 2013-
Current statusDec 4, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 13
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 13
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-1mi6
  • Surface level: 13
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1009.gif

Downloads & links

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Map

FileDownload / File: emd_1009.map.gz / Format: CCP4 / Size: 8.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThe RF2(GAQ) mutant cryo-density from the 3D-EM map of RF2 mutant bound to the E.coli 70s ribosome
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.82 Å/pix.
x 130 pix.
= 366.6 Å		2.82 Å/pix.
x 130 pix.
= 366.6 Å		2.82 Å/pix.
x 130 pix.
= 366.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.57 / Movie #1: 13
Minimum - Maximum-70.713287350000002 - 157.309265140000008
Average (Standard dev.)0.0541469 (±2.25571036)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-65-65-65
Dimensions130130130
Spacing130130130
CellA=B=C: 366.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.822.822.82
M x/y/z130130130
origin x/y/z0.0000.0000.000
length x/y/z366.600366.600366.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-65-65-65
NC/NR/NS130130130
D min/max/mean-70.713157.3090.054

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Supplemental data

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Sample components

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Entire : RF2 (GAQ) mutant from E.coli

EntireName: RF2 (GAQ) mutant from E.coli
Components
  • Sample: RF2 (GAQ) mutant from E.coli
  • Protein or peptide: RF2 (GAQ) mutant

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Supramolecule #1000: RF2 (GAQ) mutant from E.coli

SupramoleculeName: RF2 (GAQ) mutant from E.coli / type: sample / ID: 1000
Details: SPIDER was used for the computational isolation of the RF2(GAQ) mutant cryo-density from the 3D-EM map of RF2 mutant bound to the E.coli 70s ribosome (EMD-1008).
Number unique components: 1

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Macromolecule #1: RF2 (GAQ) mutant

MacromoleculeName: RF2 (GAQ) mutant / type: protein_or_peptide / ID: 1 / Recombinant expression: Yes
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

VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureAverage: 93 K
Detailsinformation is the same as in submission EMD-1008
DateAug 2, 2001
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Average electron dose: 20 e/Å2 / Details: entry EMD-1008
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 49696 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 4.05 µm / Nominal defocus min: 2.25 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Oxford, cryo-transfer 3500 / Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Detailsentry EMD-1008
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 10.9 Å
Details: information is the same as in submission 1186. SPIDER was used for the computational isolation of the RF2 (GAQ) mutant cryo-density from the 3D-EM map of RF2 mutant bound to the E.coli 70s ribosome (EMD-1008).
Number images used: 1

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Atomic model buiding 1

Initial model(PDB ID:

1m16

,

1mvr

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Output model

PDB-1mi6:
Docking of the modified RF2 X-ray structure into the Low Resolution Cryo-EM map of RF2 E.coli 70S Ribosome

PDB-1mvr:
Decoding Center & Peptidyl transferase center from the X-ray structure of the Thermus thermophilus 70S ribosome, aligned to the low resolution Cryo-EM map of E.coli 70S Ribosome

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