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- EMDB-1524: Complex of elongating Escherichia coli 70S ribosome and EF4(LepA)... -

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Basic information

Entry
Database: EMDB / ID: EMD-1524
TitleComplex of elongating Escherichia coli 70S ribosome and EF4(LepA)-GMPPNP
Map data
SampleComplex of elongating Escherichia coli 70S ribosome (2tRNAs) and EF4(LepA)-GMPPNP:
ribosome-prokaryote / EF4(LepA) / (nucleic-acidNucleic acid) x 2
Keywordsribosome / translation / LepA / EF4
Function / homology
Function and homology information


ec:3.6.5.n1: / guanosine tetraphosphate binding / ribosomal large subunit binding / stringent response / misfolded RNA binding / RNA folding / Group I intron splicing / ribosomal small subunit binding / response to salt stress / ribosomal small subunit biogenesis ...ec:3.6.5.n1: / guanosine tetraphosphate binding / ribosomal large subunit binding / stringent response / misfolded RNA binding / RNA folding / Group I intron splicing / ribosomal small subunit binding / response to salt stress / ribosomal small subunit biogenesis / translation elongation factor activity / positive regulation of RNA splicing / maintenance of translational fidelity / translational termination / response to cold / positive regulation of translation / response to pH / large ribosomal subunit rRNA binding / cytosolic large ribosomal subunit / large ribosomal subunit / ribosomal large subunit assembly / ribosome binding / cytosolic small ribosomal subunit / tRNA binding / ribosome / rRNA binding / structural constituent of ribosome / translation / GTPase activity / response to antibiotic / GTP binding / identical protein binding / plasma membrane / cytosol
Ribosomal protein L11, N-terminal / Elongation factor EFG, domain V-like / Translation elongation factor EFTu-like, domain 2 / Small GTP-binding protein domain / Ribosomal protein S12, bacterial-type / Ribosomal protein S12/S23 / Elongation factor 4 / Ribosomal protein L11, bacterial-type / Nucleic acid-binding, OB-fold / Ribosomal protein L11/L12 ...Ribosomal protein L11, N-terminal / Elongation factor EFG, domain V-like / Translation elongation factor EFTu-like, domain 2 / Small GTP-binding protein domain / Ribosomal protein S12, bacterial-type / Ribosomal protein S12/S23 / Elongation factor 4 / Ribosomal protein L11, bacterial-type / Nucleic acid-binding, OB-fold / Ribosomal protein L11/L12 / Ribosomal protein L11, C-terminal / GTP-binding protein LepA, C-terminal / Translational (tr)-type GTP-binding domain / EF-G domain III/V-like / Ribosomal protein L11, C-terminal domain superfamily / Elongation factor 4, domain IV / Ribosomal protein L11/L12, N-terminal domain superfamily / LepA, C-terminal domain superfamily / Tr-type G domain, conserved site / P-loop containing nucleoside triphosphate hydrolase / Ribosomal protein L11, conserved site
50S ribosomal protein L11 / 30S ribosomal protein S12 / Elongation factor 4
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 10.9 Å
AuthorsConnell SR / Topf M / Qin Y / Wilson DN / Mielke T / Fucini P / Nierhaus KH / Spahn CMT
CitationJournal: Nat Struct Mol Biol / Year: 2008
Title: A new tRNA intermediate revealed on the ribosome during EF4-mediated back-translocation.
Authors: Sean R Connell / Maya Topf / Yan Qin / Daniel N Wilson / Thorsten Mielke / Paola Fucini / Knud H Nierhaus / Christian M T Spahn /
Abstract: EF4 (LepA) is an almost universally conserved translational GTPase in eubacteria. It seems to be essential under environmental stress conditions and has previously been shown to back-translocate the ...EF4 (LepA) is an almost universally conserved translational GTPase in eubacteria. It seems to be essential under environmental stress conditions and has previously been shown to back-translocate the tRNAs on the ribosome, thereby reverting the canonical translocation reaction. In the current work, EF4 was directly visualized in the process of back-translocating tRNAs by single-particle cryo-EM. Using flexible fitting methods, we built a model of ribosome-bound EF4 based on the cryo-EM map and a recently published unbound EF4 X-ray structure. The cryo-EM map establishes EF4 as a noncanonical elongation factor that interacts not only with the elongating ribosome, but also with the back-translocated tRNA in the A-site region, which is present in a previously unseen, intermediate state and deviates markedly from the position of a canonical A-tRNA. Our results, therefore, provide insight into the underlying structural principles governing back-translocation.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionJun 10, 2008-
Header (metadata) releaseJun 10, 2008-
Map releaseApr 1, 2009-
UpdateApr 16, 2014-
Current statusApr 16, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3deg
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3deg
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_1524.map.gz / Format: CCP4 / Size: 12.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
2.52 Å/pix.
x 150 pix.
= 378. Å
2.52 Å/pix.
x 150 pix.
= 378. Å
2.52 Å/pix.
x 150 pix.
= 378. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.52 Å
Density
Contour LevelPrimary: 1.0 / Movie #1: 1
Minimum - Maximum-5.05751 - 11.5594
Average (Standard dev.)0.00000000080912 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-75-75-74
Dimensions150150150
Spacing150150150
CellA=B=C: 378 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.522.522.52
M x/y/z150150150
origin x/y/z0.0000.0000.000
length x/y/z378.000378.000378.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-75-75-74
NX/NY/NZ150150150
MAP C/R/S213
start NC/NR/NS-75-75-74
NC/NR/NS150150150
D min/max/mean-5.05811.5590.000

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Supplemental data

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Sample components

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Entire Complex of elongating Escherichia coli 70S ribosome (2tRNAs) and ...

EntireName: Complex of elongating Escherichia coli 70S ribosome (2tRNAs) and EF4(LepA)-GMPPNP
Number of components: 4
MassTheoretical: 2.6 MDa

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Component #1: ribosome-prokaryote, 70S ribosome

Ribosome-prokaryoteName: 70S ribosomeRibosome / a.k.a: 70S ribosomeRibosome / Prokaryote: ALL / Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli)

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Component #2: protein, EF4(LepA)

ProteinName: EF4(LepA) / Recombinant expression: Yes / Number of Copies: 1
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pET14b

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Component #3: nucleic-acid, A-tRNA

nucleic acidName: A-tRNA / a.k.a: A-tRNA / Class: T-RNA / Structure: SINGLE STRANDED / Synthetic: No
SourceSpecies: Escherichia coli (E. coli)

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Component #4: nucleic-acid, P-tRNA

nucleic acidName: P-tRNA / a.k.a: P-tRNA / Class: T-RNA / Structure: SINGLE STRANDED / Synthetic: No
SourceSpecies: Escherichia coli (E. coli)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionBuffer solution: 20 mM HEPES-KOH (pH 7.6), 4.5 mM Mg(CH3COO)2, 150 mM NH4CH3COO, 4 mM B-mercaptoethanol, 2 mM spermidine, and 0.05 mM spermine
VitrificationInstrument: FEI VITROBOT / Cryogen name: ETHANE / Humidity: 95 % / Details: Vitrification instrument: vitrobot

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
ImagingMicroscope: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 20 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 39000 X (nominal) / Imaging mode: BRIGHT FIELD / Defocus: 500 - 3900 nm
Specimen HolderHolder: Eucentric / Model: OTHER
CameraDetector: KODAK SO-163 FILM

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Image acquisition

Image acquisitionScanner: PRIMESCAN / Sampling size: 4.7 µm

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 41294 / Applied symmetry: C1 (asymmetric)
3D reconstructionSoftware: spider / CTF correction: Defocus groups / Resolution: 10.9 Å / Resolution method: FSC 0.5

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Atomic model buiding

Modeling #1Software: SITUS / Refinement protocol: rigid body / Refinement space: RECIPROCAL
Details: Protocol: Rigid Body. the subunit was fitted using situs
Input PDB model: 2i2p

2i2p
PDB Unreleased entry

Modeling #2Software: SITUS / Refinement protocol: rigid body / Refinement space: RECIPROCAL
Details: Protocol: Rigid Body. the subunit was fitted using situs
Input PDB model: 2i2t

2i2t
PDB Unreleased entry

Modeling #3Software: Mod-EM, Flex-EM / Refinement protocol: flexible / Target criteria: cross-correlation / Refinement space: REAL
Details: Protocol: Rigid Body and flexible fitting. the protein was first rigidly fit with Mod-EM and then flexibly fit with Flex-EM
Input PDB model: 3cb4
Output model

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