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Yorodumi- EMDB-1524: Complex of elongating Escherichia coli 70S ribosome and EF4(LepA)... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-1524 | |||||||||
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Title | Complex of elongating Escherichia coli 70S ribosome and EF4(LepA)-GMPPNP | |||||||||
Map data | Complex of elongating Escherichia coli 70S ribosome and EF4(LepA)-GMPPNP | |||||||||
Sample |
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Keywords | ribosome / translation / LepA / EF4 | |||||||||
Function / homology | Function and homology information : / response to pH / guanosine tetraphosphate binding / stringent response / ribosomal large subunit binding / misfolded RNA binding / Group I intron splicing / RNA folding / ribosomal small subunit binding / translation elongation factor activity ...: / response to pH / guanosine tetraphosphate binding / stringent response / ribosomal large subunit binding / misfolded RNA binding / Group I intron splicing / RNA folding / ribosomal small subunit binding / translation elongation factor activity / translational termination / response to salt stress / response to cold / positive regulation of RNA splicing / ribosomal large subunit assembly / positive regulation of translation / maintenance of translational fidelity / ribosome binding / ribosomal small subunit biogenesis / large ribosomal subunit rRNA binding / cytosolic small ribosomal subunit / cytosolic large ribosomal subunit / cytoplasmic translation / tRNA binding / rRNA binding / ribosome / structural constituent of ribosome / translation / response to antibiotic / GTPase activity / GTP binding / identical protein binding / plasma membrane / cytoplasm / cytosol Similarity search - Function | |||||||||
Biological species | Escherichia coli (E. coli) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 10.9 Å | |||||||||
Authors | Connell SR / Topf M / Qin Y / Wilson DN / Mielke T / Fucini P / Nierhaus KH / Spahn CMT | |||||||||
Citation | Journal: 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. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_1524.map.gz | 1.9 MB | EMDB map data format | |
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Header (meta data) | emd-1524-v30.xml emd-1524.xml | 11.3 KB 11.3 KB | Display Display | EMDB header |
Images | source_400_1524.gif | 59 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-1524 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-1524 | HTTPS FTP |
-Validation report
Summary document | emd_1524_validation.pdf.gz | 335.8 KB | Display | EMDB validaton report |
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Full document | emd_1524_full_validation.pdf.gz | 335.4 KB | Display | |
Data in XML | emd_1524_validation.xml.gz | 5.6 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-1524 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-1524 | HTTPS FTP |
-Related structure data
Related structure data | 3degMC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_1524.map.gz / Format: CCP4 / Size: 12.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Complex of elongating Escherichia coli 70S ribosome and EF4(LepA)-GMPPNP | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.52 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Complex of elongating Escherichia coli 70S ribosome (2tRNAs) and ...
Entire | Name: Complex of elongating Escherichia coli 70S ribosome (2tRNAs) and EF4(LepA)-GMPPNP |
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Components |
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-Supramolecule #1000: Complex of elongating Escherichia coli 70S ribosome (2tRNAs) and ...
Supramolecule | Name: Complex of elongating Escherichia coli 70S ribosome (2tRNAs) and EF4(LepA)-GMPPNP type: sample / ID: 1000 / Number unique components: 4 |
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Molecular weight | Theoretical: 2.6 MDa |
-Supramolecule #1: 70S ribosome
Supramolecule | Name: 70S ribosome / type: complex / ID: 1 / Name.synonym: 70S ribosome / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: ALL |
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Source (natural) | Organism: Escherichia coli (E. coli) |
-Macromolecule #1: EF4(LepA)
Macromolecule | Name: EF4(LepA) / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Recombinant expression: Yes |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant plasmid: pET14b |
-Macromolecule #2: A-tRNA
Macromolecule | Name: A-tRNA / type: rna / ID: 2 / Name.synonym: A-tRNA / Classification: TRANSFER / Structure: SINGLE STRANDED / Synthetic?: No |
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Source (natural) | Organism: Escherichia coli (E. coli) |
-Macromolecule #3: P-tRNA
Macromolecule | Name: P-tRNA / type: rna / ID: 3 / Name.synonym: P-tRNA / Classification: TRANSFER / Structure: SINGLE STRANDED / Synthetic?: No |
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Source (natural) | Organism: Escherichia coli (E. coli) |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | Details: 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 |
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Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Instrument: OTHER / Details: Vitrification instrument: vitrobot |
-Electron microscopy
Microscope | FEI POLARA 300 |
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Image recording | Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: PRIMESCAN / Digitization - Sampling interval: 4.7 µm / Average electron dose: 20 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.9 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 39000 |
Sample stage | Specimen holder: Eucentric / Specimen holder model: OTHER |
Experimental equipment | Model: Tecnai Polara / Image courtesy: FEI Company |
-Image processing
CTF correction | Details: Defocus groups |
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Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 10.9 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: spider / Number images used: 41294 |
-Atomic model buiding 1
Initial model | PDB ID: 2i2p |
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Software | Name: SITUS |
Details | Protocol: Rigid Body. the subunit was fitted using situs |
Refinement | Space: RECIPROCAL / Protocol: RIGID BODY FIT |
Output model | PDB-3deg: |
-Atomic model buiding 2
Initial model | PDB ID: 2i2t |
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Software | Name: SITUS |
Details | Protocol: Rigid Body. the subunit was fitted using situs |
Refinement | Space: RECIPROCAL / Protocol: RIGID BODY FIT |
Output model | PDB-3deg: |
-Atomic model buiding 3
Initial model | PDB ID: |
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Software | Name: Mod-EM and Flex-EM |
Details | Protocol: Rigid Body and flexible fitting. the protein was first rigidly fit with Mod-EM and then flexibly fit with Flex-EM |
Refinement | Space: REAL / Protocol: FLEXIBLE FIT / Target criteria: cross-correlation |
Output model | PDB-3deg: |