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Yorodumi- EMDB-3285: Cryo-electron microscopy structure of ribosome-bound initiation f... -
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-Basic information
Entry | Database: EMDB / ID: EMD-3285 | |||||||||
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Title | Cryo-electron microscopy structure of ribosome-bound initiation factor 2 | |||||||||
Map data | Initiation Factor 2 was stalled on Escherichia coli 70S ribosomes by the non-hydrolysable GTP analogue GDPNP in the presence of fMet-tRNAiMet and mRNA. | |||||||||
Sample |
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Keywords | Initiation Factor 2 / ribosome / translation / translation initiation / IF2 / translational GTPase / GTPase | |||||||||
Function / homology | Function and homology information guanosine tetraphosphate binding / stringent response / mRNA base-pairing translational repressor activity / ornithine decarboxylase inhibitor activity / ribosomal small subunit binding / misfolded RNA binding / transcription antitermination factor activity, RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation ...guanosine tetraphosphate binding / stringent response / mRNA base-pairing translational repressor activity / ornithine decarboxylase inhibitor activity / ribosomal small subunit binding / misfolded RNA binding / transcription antitermination factor activity, RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis / negative regulation of cytoplasmic translation / translational termination / chaperone-mediated protein folding / DnaA-L2 complex / four-way junction DNA binding / translation repressor activity / negative regulation of translational initiation / translational initiation / negative regulation of DNA-templated DNA replication initiation / regulation of mRNA stability / ribosome assembly / translation initiation factor activity / response to cold / mRNA regulatory element binding translation repressor activity / response to reactive oxygen species / assembly of large subunit precursor of preribosome / transcription elongation factor complex / positive regulation of RNA splicing / DNA endonuclease activity / : / cytosolic ribosome assembly / regulation of DNA-templated transcription elongation / transcription antitermination / regulation of cell growth / maintenance of translational fidelity / DNA-templated transcription termination / response to radiation / mRNA 5'-UTR binding / ribosomal small subunit biogenesis / small ribosomal subunit rRNA binding / ribosomal small subunit assembly / ribosomal large subunit assembly / cytosolic small ribosomal subunit / large ribosomal subunit rRNA binding / ribosome binding / large ribosomal subunit / ribosome biogenesis / regulation of translation / small ribosomal subunit / 5S rRNA binding / cytoplasmic translation / cytosolic large ribosomal subunit / transferase activity / negative regulation of translation / tRNA binding / molecular adaptor activity / rRNA binding / ribosome / structural constituent of ribosome / translation / response to antibiotic / mRNA binding / GTPase activity / negative regulation of DNA-templated transcription / GTP binding / DNA binding / RNA binding / zinc ion binding / membrane / cytosol / cytoplasm Similarity search - Function | |||||||||
Biological species | Escherichia coli (E. coli) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.6 Å | |||||||||
Authors | Sprink T / Ramrath DJF / Yamamoto H / Yamamoto K / Loerke J / Ismer J / Hildebrand PW / Scheerer P / Buerger J / Mielke T / Spahn CMT | |||||||||
Citation | Journal: Sci Adv / Year: 2016 Title: Structures of ribosome-bound initiation factor 2 reveal the mechanism of subunit association. Authors: Thiemo Sprink / David J F Ramrath / Hiroshi Yamamoto / Kaori Yamamoto / Justus Loerke / Jochen Ismer / Peter W Hildebrand / Patrick Scheerer / Jörg Bürger / Thorsten Mielke / Christian M T Spahn / Abstract: Throughout the four phases of protein biosynthesis-initiation, elongation, termination, and recycling-the ribosome is controlled and regulated by at least one specified translational guanosine ...Throughout the four phases of protein biosynthesis-initiation, elongation, termination, and recycling-the ribosome is controlled and regulated by at least one specified translational guanosine triphosphatase (trGTPase). Although the structural basis for trGTPase interaction with the ribosome has been solved for the last three steps of translation, the high-resolution structure for the key initiation trGTPase, initiation factor 2 (IF2), complexed with the ribosome, remains elusive. We determine the structure of IF2 complexed with a nonhydrolyzable guanosine triphosphate analog and initiator fMet-tRNAi (Met) in the context of the Escherichia coli ribosome to 3.7-Å resolution using cryo-electron microscopy. The structural analysis reveals previously unseen intrinsic conformational modes of the 70S initiation complex, establishing the mutual interplay of IF2 and initator transfer RNA (tRNA) with the ribsosome and providing the structural foundation for a mechanistic understanding of the final steps of translation initiation. | |||||||||
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_3285.map.gz | 97.9 MB | EMDB map data format | |
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Header (meta data) | emd-3285-v30.xml emd-3285.xml | 12.8 KB 12.8 KB | Display Display | EMDB header |
Images | emd_3285.png | 407.1 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-3285 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-3285 | HTTPS FTP |
-Related structure data
Related structure data | 3jcnMC 6559C 3jcjC 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_3285.map.gz / Format: CCP4 / Size: 100.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Initiation Factor 2 was stalled on Escherichia coli 70S ribosomes by the non-hydrolysable GTP analogue GDPNP in the presence of fMet-tRNAiMet and mRNA. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.23 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 : Initiation Factor 2 was stalled on Escherichia coli 70S ribosomes...
Entire | Name: Initiation Factor 2 was stalled on Escherichia coli 70S ribosomes by the non-hydrolysable GTP analogue GDPNP in the presence of fMet-tRNAiMet and mRNA. |
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Components |
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-Supramolecule #1000: Initiation Factor 2 was stalled on Escherichia coli 70S ribosomes...
Supramolecule | Name: Initiation Factor 2 was stalled on Escherichia coli 70S ribosomes by the non-hydrolysable GTP analogue GDPNP in the presence of fMet-tRNAiMet and mRNA. type: sample / ID: 1000 / Number unique components: 3 |
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Molecular weight | Experimental: 2.5 MDa / Theoretical: 2.6 MDa |
-Supramolecule #1: 70S ribosome
Supramolecule | Name: 70S ribosome / type: complex / ID: 1 / Name.synonym: 70S Details: Initiation Factor 2 was stalled on Escherichia coli 70S ribosomes by the non-hydrolysable GTP analogue GDPNP in the presence of fMet-tRNAiMet and mRNA. Recombinant expression: No / Ribosome-details: ribosome-prokaryote: ALL |
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Source (natural) | Organism: Escherichia coli (E. coli) / synonym: e.coli |
Molecular weight | Experimental: 2.5 MDa / Theoretical: 2.6 MDa |
-Macromolecule #1: fMet-tRNAiMet
Macromolecule | Name: fMet-tRNAiMet / type: rna / ID: 1 / Name.synonym: initiator tRNA Details: Initiation Factor 2 was stalled on Escherichia coli 70S ribosomes by the non-hydrolysable GTP analogue GDPNP in the presence of fMet-tRNAiMet and mRNA. Classification: TRANSFER / Structure: DOUBLE HELIX / Synthetic?: No |
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Source (natural) | Organism: Escherichia coli (E. coli) / synonym: e.coli |
-Macromolecule #2: Initiation Factor 2
Macromolecule | Name: Initiation Factor 2 / type: protein_or_peptide / ID: 2 / Name.synonym: IF2 Details: Initiation Factor 2 was stalled on Escherichia coli 70S ribosomes by the non-hydrolysable GTP analogue GDPNP in the presence of fMet-tRNAiMet and mRNA. Recombinant expression: Yes |
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Source (natural) | Organism: Escherichia coli (E. coli) / synonym: e.coli |
Molecular weight | Experimental: 97 KDa / Theoretical: 97 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant plasmid: pQE-60 |
Sequence | UniProtKB: Translation initiation factor IF-2 |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.5 Details: 20 mM Hepes-KOH pH 7.5, 15 mM magnesium acetate, 150 mM potassium acetate, 4 mM -mercapthoethanol, 2 mM spermidine and 0.05 mM spermine |
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Grid | Details: Quantifoil R3-3 Cu 300 mesh with 2 nm carbon support film |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK I / Method: blot for 2-4 seconds before plunging |
-Electron microscopy #1
Microscope | FEI POLARA 300 |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 39000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 7.18 µm / Nominal defocus min: 0.64 µm / Nominal magnification: 31000 |
Sample stage | Specimen holder model: GATAN LIQUID NITROGEN |
Microscopy ID | 1 |
Date | Aug 26, 2013 |
Image recording | Category: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number real images: 918 / Average electron dose: 20 e/Å2 / Details: Automated data collection using Leginon |
Experimental equipment | Model: Tecnai Polara / Image courtesy: FEI Company |
-Electron microscopy #2
Microscope | FEI POLARA 300 |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 39000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 7.57 µm / Nominal defocus min: 0.19 µm / Nominal magnification: 31000 |
Sample stage | Specimen holder model: GATAN LIQUID NITROGEN |
Microscopy ID | 2 |
Date | Jun 10, 2015 |
Image recording | Category: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number real images: 2797 / Average electron dose: 20 e/Å2 / Details: Automated data collection using Leginon |
Experimental equipment | Model: Tecnai Polara / Image courtesy: FEI Company |
-Image processing
CTF correction | Details: CTFFIND4 |
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Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.6 Å / Resolution method: OTHER / Software - Name: EMAN2, CTFFIND4, SPIDER, SPARX Details: Final maps were calculated from two datasets. To avoid overfitting, the data was refined in a resolution-limited scheme using SPIDER Number images used: 14872 |
Details | To avoid overfitting, the data was refined in a resolution-limited scheme using SPIDER |