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- EMDB-1671: The structure of a rabbit ribosome with an A-P' hybrid state tRNA... -

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Basic information

Entry
Database: EMDB / ID: EMD-1671
TitleThe structure of a rabbit ribosome with an A-P' hybrid state tRNA and EF-2
Map dataThis is a reconstruction of a rabbit reticulocyte ribosome stalled on a pseudoknot in an mRNA with distorted A-P' hybrid state tRNA and EF-2.
Sample
  • Sample: Rabbit reticulocyte ribosome stalled on a pseudoknot-containing mRNA with an A-P' hybrid-state tRNA and EF2
  • Organelle or cellular component: Ribosome
  • Organelle or cellular component: Elongation factor 2
  • RNA: Infectious bronchitis virus mRNA
  • RNA: tRNA
KeywordsRibosome / mRNA / tRNA / stem-loop
Biological speciesOryctolagus cuniculus (rabbit) / Infectious bronchitis virus
Methodsingle particle reconstruction / cryo EM / Resolution: 15.3 Å
AuthorsFlanagan IV JF / Namy O / Brierley I / Gilbert RJC
CitationJournal: Structure / Year: 2010
Title: Direct observation of distinct A/P hybrid-state tRNAs in translocating ribosomes.
Authors: John F Flanagan / Olivier Namy / Ian Brierley / Robert J C Gilbert /
Abstract: Transfer RNAs (tRNAs) link the genetic code in the form of messenger RNA (mRNA) to protein sequence. Translocation of tRNAs through the ribosome from aminoacyl (A) site to peptidyl (P) site and from ...Transfer RNAs (tRNAs) link the genetic code in the form of messenger RNA (mRNA) to protein sequence. Translocation of tRNAs through the ribosome from aminoacyl (A) site to peptidyl (P) site and from P site to exit site is catalyzed in eukaryotes by the translocase elongation factor 2 (EF-2) and in prokaryotes by its homolog EF-G. During tRNA movement one or more "hybrid" states (A/P) is occupied, but molecular details of them and of the translocation process are limited. Here we show by cryo-electron microscopy that a population of mammalian ribosomes stalled at an mRNA pseudoknot structure contains structurally distorted tRNAs in two different A/P hybrid states. In one (A/P'), the tRNA is in contact with the translocase EF-2, which induces it. In the other (A/P''), the translocase is absent. The existence of these alternative A/P intermediate states has relevance to our understanding of the mechanics and kinetics of translocation.
History
DepositionDec 17, 2009-
Header (metadata) releaseApr 9, 2010-
Map releaseApr 9, 2010-
UpdateApr 9, 2010-
Current statusApr 9, 2010Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 18
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 18
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-1671.gif

Downloads & links

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Map

FileDownload / File: emd_1671.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a reconstruction of a rabbit reticulocyte ribosome stalled on a pseudoknot in an mRNA with distorted A-P' hybrid state tRNA and EF-2.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.33 Å/pix.
x 128 pix.
= 426.24 Å		3.33 Å/pix.
x 128 pix.
= 426.24 Å		3.33 Å/pix.
x 128 pix.
= 426.24 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.33 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 18.0 / Movie #1: 18
Minimum - Maximum-114.019999999999996 - 202.069999999999993
Average (Standard dev.)2.38515 (±12.117599999999999)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 426.24 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.333.333.33
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z426.240426.240426.240
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-114.020202.0702.385

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Supplemental data

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Sample components

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Entire : Rabbit reticulocyte ribosome stalled on a pseudoknot-containing m...

EntireName: Rabbit reticulocyte ribosome stalled on a pseudoknot-containing mRNA with an A-P' hybrid-state tRNA and EF2
Components
  • Sample: Rabbit reticulocyte ribosome stalled on a pseudoknot-containing mRNA with an A-P' hybrid-state tRNA and EF2
  • Organelle or cellular component: Ribosome
  • Organelle or cellular component: Elongation factor 2
  • RNA: Infectious bronchitis virus mRNA
  • RNA: tRNA

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Supramolecule #1000: Rabbit reticulocyte ribosome stalled on a pseudoknot-containing m...

SupramoleculeName: Rabbit reticulocyte ribosome stalled on a pseudoknot-containing mRNA with an A-P' hybrid-state tRNA and EF2
type: sample / ID: 1000 / Oligomeric state: Ribosome, mRNA, tRNA and EF2 / Number unique components: 4

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Supramolecule #1: Ribosome

SupramoleculeName: Ribosome / type: organelle_or_cellular_component / ID: 1 / Name.synonym: 80S ribosome
Details: The sample was purified by affinity chromatography using a tag on the mRNA.
Number of copies: 1 / Oligomeric state: 40S and 60S / Recombinant expression: No
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: Rabbit / Cell: Reticulocyte

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Supramolecule #2: Elongation factor 2

SupramoleculeName: Elongation factor 2 / type: organelle_or_cellular_component / ID: 2 / Name.synonym: EF2
Details: The sample was purified by affinity chromatography using a tag on the mRNA.
Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: Rabbit / Cell: Reticulocyte

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Macromolecule #1: Infectious bronchitis virus mRNA

MacromoleculeName: Infectious bronchitis virus mRNA / type: rna / ID: 1 / Name.synonym: mRNA
Details: The sample was purified by affinity chromatography.
Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: Infectious bronchitis virus

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Macromolecule #2: tRNA

MacromoleculeName: tRNA / type: rna / ID: 2 / Name.synonym: tRNA
Details: The sample was purified by affinity chromatography using a tag on the mRNA.
Classification: TRANSFER / Structure: OTHER / Synthetic?: No
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: Rabbit

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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: 25mM Hepes-KOH pH 7.8, 5mM MgOAc, 1mM cycloheximide, 150mM KOAc
GridDetails: 300 mesh copper grid with lacey carbon film
VitrificationCryogen name: ETHANE / Chamber temperature: 100 K / Instrument: HOMEMADE PLUNGER / Details: Vitrification instrument: Homemade plunger / Method: Blot with Whatman number 1 paper

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Electron microscopy

MicroscopeFEI TECNAI F30
TemperatureAverage: 115 K
Alignment procedureLegacy - Astigmatism: At 200,000 magnification
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 107 / Bits/pixel: 8
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 5.3 µm / Nominal defocus min: 0.92 µm / Nominal magnification: 38000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Per micrograph
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 15.3 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Spider, WellMAP
Details: Maps were calculated using 20215 out of 64413 particles selected by the A-P-state tRNA and EF2 presence. Presence of the tRNA was proved using multivariate statistical analysis focused ...Details: Maps were calculated using 20215 out of 64413 particles selected by the A-P-state tRNA and EF2 presence. Presence of the tRNA was proved using multivariate statistical analysis focused classification of particles without previous alignment to a tRNA-containing ribosome.
Number images used: 20215
Final angle assignmentDetails: Spider convention

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Atomic model buiding 1

Initial modelPDB ID:

2zkq
PDB Unreleased entry

SoftwareName: URO
DetailsProtocol: Rigid body in segments. Subregions were defined as described in the paper accompanying this deposition.
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient

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Atomic model buiding 2

Initial modelPDB ID:

1evv

SoftwareName: URO, Refmac, NMFF
DetailsProtocol: Rigid body, Refmac and normal modes fitting. The tRNA crystal structure was regularized in Refmac (CCP4) and then subjected to normal modes-assisted flexible fitting using the program NMFF (Tama et al.)
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 3

Initial modelPDB ID:

1s1h
PDB Unreleased entry

SoftwareName: URO, Refmac, NMFF
DetailsProtocol: Rigid body, Refmac and normal modes fitting
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 4

Initial modelPDB ID:

2zkr
PDB Unreleased entry

SoftwareName: URO
DetailsProtocol: Rigid body in segments. Subregions were defined as described in the paper accompanying this deposition.
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient

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