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- EMDB-1184: Interactions of the release factor RF1 with the ribosome as revea... -

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Basic information

Entry
Database: EMDB / ID: EMD-1184
TitleInteractions of the release factor RF1 with the ribosome as revealed by cryo-EM.
Map dataThis is an em map of a 70s E.coli ribosome
Sample
  • Sample: Release complex bound with RF1-WT
  • Complex: 30S
  • Complex: 50S
  • RNA: P-site tRNA
  • RNA: E-site tRNA
  • RNA: mRNA
  • Protein or peptide: RF1
Function / homology
Function and homology information


translation release factor activity, codon specific / cytoplasm
Similarity search - Function
Peptide chain release factor 1 / : / 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
Similarity search - Domain/homology
Peptide chain release factor 1
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 12.8 Å
AuthorsRawat U / Gao H / Zavialov A / Gursky R / Ehrenberg M / Frank J
CitationJournal: J Mol Biol / Year: 2006
Title: Interactions of the release factor RF1 with the ribosome as revealed by cryo-EM.
Authors: Urmila Rawat / Haixiao Gao / Andrey Zavialov / Richard Gursky / Måns Ehrenberg / Joachim Frank /
Abstract: In eubacteria, termination of translation is signaled by any one of the stop codons UAA, UAG, and UGA moving into the ribosomal A site. Two release factors, RF1 and RF2, recognize and bind to the ...In eubacteria, termination of translation is signaled by any one of the stop codons UAA, UAG, and UGA moving into the ribosomal A site. Two release factors, RF1 and RF2, recognize and bind to the stop codons with different affinities and trigger the hydrolysis of the ester bond that links the polypeptide with the P-site tRNA. Cryo-electron microscopy (cryo-EM) results obtained in this study show that ribosome-bound RF1 is in an open conformation, unlike the closed conformation observed in the crystal structure of the free factor, allowing its simultaneous access to both the decoding center and the peptidyl-transferase center. These results are similar to those obtained for RF2, but there is an important difference in how the factors bind to protein L11, which forms part of the GTPase-associated center of the large ribosomal subunit. The difference in the binding position, C-terminal domain for RF2 versus N-terminal domain for RF1, explains a body of L11 mutation studies that revealed differential effects on the activity of the two factors. Very recent data obtained with small-angle X-ray scattering now reveal that the solution structure of RF1 is open, as here seen on the ribosome by cryo-EM, and not closed, as seen in the crystal.
History
DepositionJan 31, 2006-
Header (metadata) releaseJan 31, 2006-
Map releaseMay 9, 2006-
UpdateJun 29, 2016-
Current statusJun 29, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 20
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 20
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-2fvo, PDB-3dg4
  • Surface level: 20
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-2fvo
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1184.gif

Downloads & links

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Map

FileDownload / File: emd_1184.map.gz / Format: CCP4 / Size: 8.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is an em map of a 70s E.coli 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 Level1: 50.799999999999997 / Movie #1: 20
Minimum - Maximum-115.182000000000002 - 225.712999999999994
Average (Standard dev.)4.81105 (±21.584599999999998)
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-115.182225.7134.811

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Supplemental data

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Sample components

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Entire : Release complex bound with RF1-WT

EntireName: Release complex bound with RF1-WT
Components
  • Sample: Release complex bound with RF1-WT
  • Complex: 30S
  • Complex: 50S
  • RNA: P-site tRNA
  • RNA: E-site tRNA
  • RNA: mRNA
  • Protein or peptide: RF1

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Supramolecule #1000: Release complex bound with RF1-WT

SupramoleculeName: Release complex bound with RF1-WT / type: sample / ID: 1000 / Number unique components: 6

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Supramolecule #1: 30S

SupramoleculeName: 30S / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: SSU 30S
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MRE600

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Supramolecule #2: 50S

SupramoleculeName: 50S / type: complex / ID: 2 / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: LSU 50S
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MRE600

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Macromolecule #1: P-site tRNA

MacromoleculeName: P-site tRNA / type: rna / ID: 1 / Classification: OTHER / Structure: OTHER / Synthetic?: No
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #2: E-site tRNA

MacromoleculeName: E-site tRNA / type: rna / ID: 2 / Classification: OTHER / Structure: OTHER / Synthetic?: No
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #3: mRNA

MacromoleculeName: mRNA / type: rna / ID: 3 / Classification: OTHER / Structure: DOUBLE HELIX / Synthetic?: Yes
Source (natural)Organism: synthetic construct (others)

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Macromolecule #4: RF1

MacromoleculeName: RF1 / type: protein_or_peptide / ID: 4 / Recombinant expression: No
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MRE600

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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 / Chamber humidity: 90 % / Chamber temperature: 93 K / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: two sided blotting plunger
Method: Blot for 2 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureAverage: 93 K
Alignment procedureLegacy - Electron beam tilt params: 0
DateDec 1, 2002
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 14 µm / Number real images: 56 / Average electron dose: 20 e/Å2 / Od range: 1.2 / 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 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Cryo stage / Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: defocus groups
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 12.8 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER, package / Number images used: 24622
Final angle assignmentDetails: SPIDER: theta 15 degrees, phi 15 degrees

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Atomic model buiding 1

Initial modelPDB ID:

1rq0


Chain - Chain ID: A
SoftwareName: O
DetailsProtocol: Rigid Body. manual fitting in O
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: correlation coefficient
Output model

PDB-2fvo:
Docking of the modified RF1 X-ray structure into the Low Resolution Cryo-EM map of E.coli 70S Ribosome bound with RF1

PDB-3dg4:
Coordinates of 16S and 23S rRNAs fitted into the cryo-EM map of RF1-bound termination complex

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