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Entry
Database: EMDB / ID: EMD-6559
TitleCryo-electron microscopy structure of ribosome-bound initiation factor 2 70S IC II state
Map dataReconstruction of the 70S-fMet-tRNAiMet-IF2-GDPNP complex, IC II state
Sample
  • Sample: Escherichia coli Initiation Factor 2 stalled on Escherichia coli 70S ribosomes by the non-hydrolysable GTP analogue GDPNP in the presence of fMet-tRNAiMet and mRNA
  • Complex: 70S ribosomeRibosome
  • RNA: fMet-tRNAiMet
  • Protein or peptide: Initiation Factor 2
Keywordstranslation initiation / ribosome / initiation / Initiation Factor 2 / 70S initiation complex / prokaryotes / GTPase / translation 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 / negative regulation of translational initiation / translation repressor activity / 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 / positive regulation of RNA splicing / DNA endonuclease activity / transcription elongation factor complex / 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 / cytoplasmic translation / small ribosomal subunit / 5S rRNA binding / cytosolic large ribosomal subunit / transferase activity / tRNA binding / negative regulation of translation / rRNA binding / molecular adaptor activity / 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
Initiation factor 2 associated domain, bacterial / Bacterial translation initiation factor IF-2 associated region / Translation initiation factor IF-2, N-terminal / Translation initiation factor IF-2, N-terminal region / Translation initiation factor IF-2, domain II / Initiation factor 2 signature. / Translation initiation factor IF-2, bacterial-like / Translation initiation factor IF- 2, domain 3 / Translation-initiation factor 2 / Translation initiation factor IF- 2 ...Initiation factor 2 associated domain, bacterial / Bacterial translation initiation factor IF-2 associated region / Translation initiation factor IF-2, N-terminal / Translation initiation factor IF-2, N-terminal region / Translation initiation factor IF-2, domain II / Initiation factor 2 signature. / Translation initiation factor IF-2, bacterial-like / Translation initiation factor IF- 2, domain 3 / Translation-initiation factor 2 / Translation initiation factor IF- 2 / Translation initiation factor IF-2, domain 3 superfamily / Putative DNA-binding domain superfamily / Ribosomal protein L10, eubacterial, conserved site / Ribosomal protein L10 signature. / Ribosomal protein L10 / : / Translation elongation factor EFTu-like, domain 2 / Ribosomal protein S21, conserved site / Ribosomal protein S21 signature. / Ribosomal protein L25, short-form / Elongation factor Tu domain 2 / Ribosomal protein S14, bacterial/plastid / Ribosomal protein L11, bacterial-type / Ribosomal protein S21 superfamily / Ribosomal protein S21 / Ribosomal protein S16, conserved site / Ribosomal protein S16 signature. / Translational (tr)-type GTP-binding domain / Elongation factor Tu GTP binding domain / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / Ribosomal protein S21 / Ribosomal protein L11, conserved site / Ribosomal protein L10-like domain superfamily / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / Ribosomal protein L10P / Ribosomal protein L10 / Ribosomal protein L11 signature. / Ribosomal protein L16 signature 1. / : / Ribosomal protein L6, conserved site / Ribosomal protein L6 signature 1. / Ribosomal protein L16, conserved site / Ribosomal protein L16 signature 2. / Ribosomal protein L11, N-terminal / Ribosomal protein L17 signature. / Ribosomal protein L9 signature. / Ribosomal protein L9, bacteria/chloroplast / Ribosomal protein L9, C-terminal / Ribosomal protein L9, C-terminal domain / Ribosomal protein L9, C-terminal domain superfamily / Ribosomal protein L11/L12 / Ribosomal protein L11, C-terminal / Ribosomal protein L11, C-terminal domain superfamily / Ribosomal protein L11/L12, N-terminal domain superfamily / Ribosomal protein L11/L12 / Ribosomal protein L11, N-terminal domain / Ribosomal protein L11, RNA binding domain / Ribosomal L25p family / Ribosomal protein L25 / Ribosomal protein L28/L24 superfamily / Ribosomal protein L36 signature. / Ribosomal protein L25/Gln-tRNA synthetase, N-terminal / Ribosomal protein L32p, bacterial type / Ribosomal protein L25/Gln-tRNA synthetase, anti-codon-binding domain superfamily / Ribosomal protein L9, N-terminal domain superfamily / Ribosomal protein L9 / Ribosomal protein L9, N-terminal / Ribosomal protein L9, N-terminal domain / Ribosomal protein L28 / Ribosomal protein L35, conserved site / Ribosomal protein L35 signature. / Ribosomal protein L33, conserved site / Ribosomal protein L33 signature. / Ribosomal protein L35, non-mitochondrial / Ribosomal protein L5, bacterial-type / Ribosomal protein L6, bacterial-type / Ribosomal protein L18, bacterial-type / Ribosomal protein L19, conserved site / Ribosomal protein L19 signature. / Ribosomal protein L36 / Ribosomal protein L36 superfamily / Ribosomal protein L36 / Ribosomal protein L9/RNase H1, N-terminal / Ribosomal protein L20 signature. / Ribosomal protein S3, bacterial-type / Ribosomal protein S6, conserved site / Ribosomal protein S6 signature. / Ribosomal protein L27, conserved site / Ribosomal protein L27 signature. / Ribosomal protein S19, bacterial-type / Ribosomal protein S7, bacterial/organellar-type / Ribosomal protein S11, bacterial-type / Ribosomal protein S13, bacterial-type / Ribosomal protein S20 / Ribosomal protein S20 superfamily / Ribosomal protein S20 / Ribosomal protein S9, bacterial/plastid / Ribosomal protein S4, bacterial-type / Ribosomal protein L14P, bacterial-type
Similarity search - Domain/homology
Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL15 / Translation initiation factor IF-2 / Large ribosomal subunit protein uL10 / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL28 ...Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL15 / Translation initiation factor IF-2 / Large ribosomal subunit protein uL10 / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL28 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein bL32 / Large ribosomal subunit protein bL33 / Large ribosomal subunit protein bL34 / Large ribosomal subunit protein bL35 / Large ribosomal subunit protein bL36A / Large ribosomal subunit protein bL9 / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein uS11 / Small ribosomal subunit protein uS12 / Small ribosomal subunit protein uS13 / Small ribosomal subunit protein bS16 / Small ribosomal subunit protein bS18 / Small ribosomal subunit protein uS19 / Small ribosomal subunit protein bS20 / Small ribosomal subunit protein uS2 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS4 / Small ribosomal subunit protein uS5 / Small ribosomal subunit protein uS8 / Small ribosomal subunit protein uS9 / Large ribosomal subunit protein uL13 / Large ribosomal subunit protein uL14 / Large ribosomal subunit protein uL16 / Large ribosomal subunit protein uL23 / Small ribosomal subunit protein uS15 / Large ribosomal subunit protein bL17 / Large ribosomal subunit protein bL21 / Large ribosomal subunit protein uL30 / Large ribosomal subunit protein uL6 / Small ribosomal subunit protein uS14 / Small ribosomal subunit protein uS17 / Large ribosomal subunit protein uL18 / Large ribosomal subunit protein uL2 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein uL24 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL22 / Large ribosomal subunit protein uL5 / Small ribosomal subunit protein bS21 / Large ribosomal subunit protein bL25
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsSprink T / Ramrath DJF / Yamamoto H / Yamamoto K / Loerke J / Ismer J / Hildebrand PW / Scheerer P / Buerger J / Mielke T / Spahn CMT
CitationJournal: 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
DepositionDec 17, 2015-
Header (metadata) releaseFeb 17, 2016-
Map releaseMar 9, 2016-
UpdateMar 9, 2016-
Current statusMar 9, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.2
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 2.2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jcj
  • Surface level: 2.2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jcj
  • Surface level: 2.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6559.png

Downloads & links

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Map

FileDownload / File: emd_6559.map.gz / Format: CCP4 / Size: 173.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the 70S-fMet-tRNAiMet-IF2-GDPNP complex, IC II state
Voxel sizeX=Y=Z: 1.025 Å
Density
Contour LevelBy AUTHOR: 2.2 / Movie #1: 2.2
Minimum - Maximum-6.02793121 - 13.34954739
Average (Standard dev.)0.09076034 (±0.65751249)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 369.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0251.0251.025
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z369.000369.000369.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-6.02813.3500.091

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Supplemental data

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Sample components

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Entire : Escherichia coli Initiation Factor 2 stalled on Escherichia coli ...

EntireName: Escherichia coli Initiation Factor 2 stalled on Escherichia coli 70S ribosomes by the non-hydrolysable GTP analogue GDPNP in the presence of fMet-tRNAiMet and mRNA
Components
  • Sample: Escherichia coli Initiation Factor 2 stalled on Escherichia coli 70S ribosomes by the non-hydrolysable GTP analogue GDPNP in the presence of fMet-tRNAiMet and mRNA
  • Complex: 70S ribosomeRibosome
  • RNA: fMet-tRNAiMet
  • Protein or peptide: Initiation Factor 2

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Supramolecule #1000: Escherichia coli Initiation Factor 2 stalled on Escherichia coli ...

SupramoleculeName: Escherichia coli Initiation Factor 2 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
Molecular weightExperimental: 2.5 MDa / Theoretical: 2.6 MDa

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Supramolecule #1: 70S ribosome

SupramoleculeName: 70S ribosome / type: complex / ID: 1 / Name.synonym: 70S / Recombinant expression: No / Database: NCBI / Ribosome-details: ribosome-prokaryote: ALL
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightExperimental: 2.5 MDa / Theoretical: 2.6 MDa

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Macromolecule #1: fMet-tRNAiMet

MacromoleculeName: fMet-tRNAiMet / type: rna / ID: 1 / Name.synonym: initiator tRNA / Classification: TRANSFER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #2: Initiation Factor 2

MacromoleculeName: Initiation Factor 2 / type: protein_or_peptide / ID: 2 / Name.synonym: IF2 / Details: bound to GNPPNP / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightExperimental: 97 KDa / Theoretical: 97 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pQE-60
SequenceUniProtKB: Translation initiation factor IF-2

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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: 7.5
Details: 20 mM HEPES-KOH, pH 7.5, 15 mM magnesium acetate, 150 mM potassium acetate, 4 mM 2-mercapthoethanol, 2 mM spermidine, 0.05 mM spermine
GridDetails: Quantifoil R3-3 Cu 300 mesh with 2 nm carbon support film
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK I / Method: Blot for 2-4 seconds before plunging.

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Electron microscopy #1

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated 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 stageSpecimen holder model: GATAN LIQUID NITROGEN
Microscopy ID1
DateAug 26, 2013
Image recordingCategory: 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

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Electron microscopy #2

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated 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 stageSpecimen holder model: GATAN LIQUID NITROGEN
Microscopy ID2
DateJun 10, 2015
Image recordingCategory: 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

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Image processing

CTF correctionDetails: CTFFIND4
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: OTHER / Software - Name: EMAN2, CTFFIND4, SPIDER, SPARX
Details: Final maps were calculated from two combined datasets. To avoid overfitting, the data were refined in a resolution-limited scheme using SPIDER.
Number images used: 54585
DetailsTo avoid overfitting, the data was refined in a resolution-limited scheme using SPIDER.

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