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Basic information

Entry
Database: EMDB / ID: EMD-1005
TitleStructure of the Escherichia coli ribosomal termination complex with release factor 2.
Map dataE. coli ribosomal termination complex with release factor RF2.
Sample
  • Sample: release factor RF2 bound to E. coli ribosomes
  • Complex: ribosome
  • Protein or peptide: release factor 2
Function / homology
Function and homology information


translation release factor activity, codon specific / translational termination / response to radiation / ribosomal large subunit assembly / large ribosomal subunit rRNA binding / large ribosomal subunit / regulation of translation / small ribosomal subunit / 5S rRNA binding / cytosolic large ribosomal subunit ...translation release factor activity, codon specific / translational termination / response to radiation / ribosomal large subunit assembly / large ribosomal subunit rRNA binding / large ribosomal subunit / regulation of translation / small ribosomal subunit / 5S rRNA binding / cytosolic large ribosomal subunit / cytoplasmic translation / transferase activity / tRNA binding / negative regulation of translation / rRNA binding / ribosome / structural constituent of ribosome / translation / ribonucleoprotein complex / mRNA binding / zinc ion binding / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Peptide chain release factor 2 / Peptide chain release factor 2 / Ribosomal protein L10e, archaea / Ribosomal protein L3, archaeal / Ribosomal protein L4, archaea / Ribosomal protein L5, archaeal / Peptide chain release factor / PCRF domain / PCRF / Peptide chain release factor class I superfamily ...Peptide chain release factor 2 / Peptide chain release factor 2 / Ribosomal protein L10e, archaea / Ribosomal protein L3, archaeal / Ribosomal protein L4, archaea / Ribosomal protein L5, archaeal / Peptide chain release factor / PCRF domain / PCRF / Peptide chain release factor class I superfamily / Prokaryotic-type class I peptide chain release factors signature. / Ribosomal protein L24e / Peptide chain release factor class I / RF-1 domain / Ribosomal L15/L27a, N-terminal / Ribosomal protein L23 / Ribosomal protein L1, bacterial-type / 30S ribosomal protein Thx / 30S ribosomal protein Thx / 30S ribosomal protein / metallochaperone-like domain / TRASH domain / Ribosomal protein L10e, conserved site / Ribosomal protein L10e / Ribosomal protein L24e, conserved site / Ribosomal protein S14, type Z / Ribosomal protein L1, conserved site / Ribosomal protein L1 / Ribosomal protein L1 signature. / Ribosomal protein L10e signature. / Ribosomal protein L1, 3-layer alpha/beta-sandwich / Ribosomal protein L24e signature. / Ribosomal protein L4/L1e, eukaryotic/archaeal, conserved site / Ribosomal protein L3, domain 3, archaeal type superfamily / Ribosomal protein L3, archaeal/eukaryotic type / Ribosomal protein L24e-related / Ribosomal protein L24e/L24 superfamily / Ribosomal protein L26/L24, eukaryotic/archaeal / Ribosomal protein L25, short-form / Ribosomal protein L13, eukaryotic/archaeal / Ribosomal protein L4, eukaryotic and archaeal type / Ribosomal protein L1-like / Ribosomal protein L1/ribosomal biogenesis protein / Ribosomal protein L24e / Ribosomal protein L1p/L10e family / Ribosomal proteins L26 eukaryotic, L24P archaeal / Ribosomal protein L5 eukaryotic/L18 archaeal / Ribosomal large subunit proteins 60S L5, and 50S L18 / Ribosomal protein L11, bacterial-type / Ribosomal protein L1e signature. / Ribosomal protein L11, conserved site / Ribosomal protein L11 signature. / Ribosomal protein L6, conserved site / Ribosomal protein L6 signature 1. / Ribosomal protein L11, N-terminal / 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, N-terminal domain / Ribosomal protein L11, RNA binding domain / Ribosomal L25p family / Ribosomal protein L25 / Ribosomal protein S14/S29 / Ribosomal protein L25/Gln-tRNA synthetase, N-terminal / Ribosomal protein L25/Gln-tRNA synthetase, anti-codon-binding domain superfamily / Ribosomal protein L6, bacterial-type / Ribosomal protein S3, bacterial-type / Ribosomal protein S6, conserved site / Ribosomal protein S6 signature. / Ribosomal protein S19, bacterial-type / Ribosomal protein S7, bacterial/organellar-type / Ribosomal protein S11, bacterial-type / Ribosomal protein S13, bacterial-type / Ribosomal protein S20 / Ribosomal protein S20 superfamily / Ribosomal protein S20 / Ribosomal protein S9, bacterial/plastid / Ribosomal protein L14P, bacterial-type / Ribosomal protein S4, bacterial-type / 30S ribosomal protein S17 / Ribosomal protein S5, bacterial-type / Ribosomal protein L22, bacterial/chloroplast-type / Ribosomal protein S6, plastid/chloroplast / Ribosomal protein L2, bacterial/organellar-type / Ribosomal protein S2, bacteria/mitochondria/plastid / : / Ribosomal protein L30, bacterial-type / Ribosomal protein S18, conserved site / Ribosomal protein S18 signature. / Ribosomal protein S16 / Ribosomal protein S16 / Ribosomal protein S16 domain superfamily / Ribosomal protein S15, bacterial-type / Ribosomal protein S6 / Ribosomal protein S6 / Ribosomal protein S6 superfamily / Ribosomal protein S12, bacterial-type / Translation elongation factor EF1B/ribosomal protein S6
Similarity search - Domain/homology
Large ribosomal subunit protein uL6 / Large ribosomal subunit protein uL14 / Peptide chain release factor RF2 / Small ribosomal subunit protein uS14 / Small ribosomal subunit protein uS17 / Small ribosomal subunit protein uS8 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein uL24 / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein uL4 ...Large ribosomal subunit protein uL6 / Large ribosomal subunit protein uL14 / Peptide chain release factor RF2 / Small ribosomal subunit protein uS14 / Small ribosomal subunit protein uS17 / Small ribosomal subunit protein uS8 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein uL24 / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein eL24 / Large ribosomal subunit protein uL18 / Large ribosomal subunit protein uL5 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein uL13 / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein uL16 / Large ribosomal subunit protein bL25 / Small ribosomal subunit protein uS2 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS4 / Small ribosomal subunit protein uS9 / Small ribosomal subunit protein uS11 / Small ribosomal subunit protein uS13 / Small ribosomal subunit protein bS20 / Large ribosomal subunit protein uL2 / Small ribosomal subunit protein uS12 / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein uS19 / Large ribosomal subunit protein uL22 / Small ribosomal subunit protein uS5 / Large ribosomal subunit protein uL30 / Small ribosomal subunit protein bTHX / Small ribosomal subunit protein uS15 / Small ribosomal subunit protein bS16 / Large ribosomal subunit protein uL1 / Small ribosomal subunit protein bS6 / Small ribosomal subunit protein bS18
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Escherichia coli BL21(DE3) (bacteria)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 14.0 Å
AuthorsKlaholz BP / Pape T / Zavialov AV / Myasnikov AG / Orlova EV / Vestergaard B / Ehrenberg M / van Heel M
CitationJournal: Nature / Year: 2003
Title: Structure of the Escherichia coli ribosomal termination complex with release factor 2.
Authors: Bruno P Klaholz / Tillmann Pape / Andrey V Zavialov / Alexander G Myasnikov / Elena V Orlova / Bente Vestergaard / Måns Ehrenberg / Marin van Heel /
Abstract: Termination of protein synthesis occurs when the messenger RNA presents a stop codon in the ribosomal aminoacyl (A) site. Class I release factor proteins (RF1 or RF2) are believed to recognize stop ...Termination of protein synthesis occurs when the messenger RNA presents a stop codon in the ribosomal aminoacyl (A) site. Class I release factor proteins (RF1 or RF2) are believed to recognize stop codons via tripeptide motifs, leading to release of the completed polypeptide chain from its covalent attachment to transfer RNA in the ribosomal peptidyl (P) site. Class I RFs possess a conserved GGQ amino-acid motif that is thought to be involved directly in protein-transfer-RNA bond hydrolysis. Crystal structures of bacterial and eukaryotic class I RFs have been determined, but the mechanism of stop codon recognition and peptidyl-tRNA hydrolysis remains unclear. Here we present the structure of the Escherichia coli ribosome in a post-termination complex with RF2, obtained by single-particle cryo-electron microscopy (cryo-EM). Fitting the known 70S and RF2 structures into the electron density map reveals that RF2 adopts a different conformation on the ribosome when compared with the crystal structure of the isolated protein. The amino-terminal helical domain of RF2 contacts the factor-binding site of the ribosome, the 'SPF' loop of the protein is situated close to the mRNA, and the GGQ-containing domain of RF2 interacts with the peptidyl-transferase centre (PTC). By connecting the ribosomal decoding centre with the PTC, RF2 functionally mimics a tRNA molecule in the A site. Translational termination in eukaryotes is likely to be based on a similar mechanism.
History
DepositionAug 28, 2002-
Header (metadata) releaseAug 30, 2002-
Map releaseAug 30, 2003-
UpdateOct 22, 2014-
Current statusOct 22, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.028
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.028
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-1ml5
  • Surface level: 0.028
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1005.gif

Downloads & links

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Map

FileDownload / File: emd_1005.map.gz / Format: CCP4 / Size: 6.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationE. coli ribosomal termination complex with release factor RF2.
Voxel sizeX=Y=Z: 2.419 Å
Density
Contour Level1: 0.0401 / Movie #1: 0.028
Minimum - Maximum-0.159669 - 0.213811
Average (Standard dev.)-0.000130684 (±0.0224271)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-60-60-60
Dimensions120120120
Spacing120120120
CellA=B=C: 290.28 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.4192.4192.419
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z290.280290.280290.280
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-60-60-60
NC/NR/NS120120120
D min/max/mean-0.1600.214-0.000

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Supplemental data

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Sample components

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Entire : release factor RF2 bound to E. coli ribosomes

EntireName: release factor RF2 bound to E. coli ribosomes
Components
  • Sample: release factor RF2 bound to E. coli ribosomes
  • Complex: ribosome
  • Protein or peptide: release factor 2

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Supramolecule #1000: release factor RF2 bound to E. coli ribosomes

SupramoleculeName: release factor RF2 bound to E. coli ribosomes / type: sample / ID: 1000 / Oligomeric state: monomer / Number unique components: 2
Molecular weightExperimental: 2.3 MDa / Method: sedimentation

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Supramolecule #1: ribosome

SupramoleculeName: ribosome / type: complex / ID: 1 / Name.synonym: ribosome
Details: Proteins L7/L12 of the LSU 50S subunit are not seen in the map
Recombinant expression: No / Ribosome-details: ribosome-prokaryote: LSU 50S
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightExperimental: 2.3 MDa / Theoretical: 2.3 MDa

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Macromolecule #1: release factor 2

MacromoleculeName: release factor 2 / type: protein_or_peptide / ID: 1 / Name.synonym: RF2 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21 (DE3)
Molecular weightExperimental: 40 KDa / Theoretical: 40 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET11a
SequenceInterPro: Peptide chain release factor 2

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.15 mg/mL
BufferpH: 7.5 / Details: polymix buffer
StainingType: NEGATIVE / Details: no staining, cryo-EM with holey carbon grids
VitrificationCryogen name: ETHANE / Chamber humidity: 45 % / Chamber temperature: 20 K / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: home-made cryo-plunger / Method: blot for 2 seconds before plunging

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG/ST
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 48000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.1 mm / Nominal defocus max: 2.9 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Side entry liquid nitrogen-cooled cryo specimen holder.
Specimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 100 K / Max: 102 K / Average: 100 K
DateSep 6, 2001
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: PATCHWORK DENSITOMETER / Digitization - Sampling interval: 3 µm / Number real images: 78 / Average electron dose: 10 e/Å2 / Bits/pixel: 12

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Image processing

CTF correctionDetails: each particle
Final two d classificationNumber classes: 103
Final angle assignmentDetails: beta gamma
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 14.0 Å / Resolution method: OTHER / Software - Name: IMAGIC / Details: exact filtered back-projection / Number images used: 15800

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Atomic model buiding 1

Initial modelPDB ID:

1gix
PDB Unreleased entry

SoftwareName: eye
DetailsProtocol: rigid body. rigid body of three individual domains keeping the connectivity; linkers betweenn domains defined based on temperature factors of crystal structure, conserved Gly and Pro residues, proteolytic sites, and global domain architecture
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: best visual fit using the program O
Output model

PDB-1ml5:
Structure of the E. coli ribosomal termination complex with release factor 2

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Atomic model buiding 2

Initial modelPDB ID:

1giy
PDB Unreleased entry

SoftwareName: eye
DetailsProtocol: rigid body. rigid body of three individual domains keeping the connectivity; linkers betweenn domains defined based on temperature factors of crystal structure, conserved Gly and Pro residues, proteolytic sites, and global domain architecture
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: best visual fit using the program O
Output model

PDB-1ml5:
Structure of the E. coli ribosomal termination complex with release factor 2

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