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- EMDB-1006: A cryo-electron microscopic study of ribosome-bound termination f... -

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Basic information

Entry
Database: EMDB / ID: EMD-1006
TitleA cryo-electron microscopic study of ribosome-bound termination factor RF2.
Map dataRibosome-bound termination factor RF2
Sample
  • Sample: E.coli 70s ribosome
  • Complex: E.coli 70s ribosome
  • Ligand: peptidyl P-tRNA
  • Ligand: E-tRNA
  • Ligand: MFTI-mRNA
Function / homology
Function and homology information


translation release factor activity, codon specific / translational termination / small ribosomal subunit / large ribosomal subunit rRNA binding / 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: 11.3 Å
AuthorsRawat U / Gao H / Zavialov A / Gursky R / 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 1, 2002-
Header (metadata) releaseSep 26, 2002-
Map releaseSep 26, 2003-
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: 30
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 30
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-1mi6, PDB-1mvr
  • Surface level: 30
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-1mi6
  • Imaged by Jmol
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-1mvr
  • Imaged by Jmol
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1006.gif

Downloads & links

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Map

FileDownload / File: emd_1006.map.gz / Format: CCP4 / Size: 8.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRibosome-bound termination factor RF2
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 Level1: 48.100000000000001 / Movie #1: 30
Minimum - Maximum-144.453000000000003 - 251.236999999999995
Average (Standard dev.)2.32248 (±24.480699999999999)
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 °

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-144.453251.2372.322

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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: E.coli 70s ribosome
  • Ligand: peptidyl P-tRNA
  • Ligand: E-tRNA
  • Ligand: MFTI-mRNA

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Supramolecule #1000: E.coli 70s ribosome

SupramoleculeName: E.coli 70s ribosome / type: sample / ID: 1000 / Number unique components: 4
Molecular weightTheoretical: 2.5 MDa

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Supramolecule #1: E.coli 70s ribosome

SupramoleculeName: E.coli 70s ribosome / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: LSU 50S
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 2.5 MDa

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Macromolecule #1: peptidyl P-tRNA

MacromoleculeName: peptidyl P-tRNA / type: ligand / ID: 1 / Recombinant expression: No
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #2: E-tRNA

MacromoleculeName: E-tRNA / type: ligand / ID: 2 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #3: MFTI-mRNA

MacromoleculeName: MFTI-mRNA / type: ligand / ID: 3
Details: Zavialov et al., A posttermination ribosomal complex is the guanine nucleotide exchange factor for peptide release factor RF3. Cell. 107,1-20 (2001).
Number of copies: 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

Concentration0.09 mg/mL
BufferpH: 7.5
Details: Polymix buffer, containing at final concentration 5 mM potassium phosphate, 5 mM magnesium acetate, 5mM ammonium chloride, 95 mM potassium chloride, 0.5 mM calcium chloride, 8mM putrescine, 1mM dithioerythritol
VitrificationCryogen name: ETHANE / Chamber humidity: 58 % / Chamber temperature: 36 K / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: Two side blotting plunger. Rapid-freezing in liquid ethane
Method: Blot and Plunge

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureAverage: 93 K
DateAug 9, 2001
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 14 µm / Number real images: 34 / Average electron dose: 20 e/Å2 / Bits/pixel: 12
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: 5.975 µm / Nominal defocus min: 1.39 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

CTF correctionDetails: Wiener filtration of defocus groups
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 11.3 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER/WEB
Details: Frank, J. (2000) Three-Dimensional Cryoelectron Microscopy of Ribosomes, Methods of Enzymology (Ch.18) 317, 276-291.
Number images used: 14965

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Atomic model buiding 1

Initial model(PDB ID:

1m16

,

1mvr

)
Detailsmanual fitting using O
RefinementProtocol: RIGID BODY FIT
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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