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Entry
Database: EMDB / ID: EMD-3285
TitleCryo-electron microscopy structure of ribosome-bound initiation factor 2
Map dataInitiation 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
  • Sample: 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.
  • Complex: 70S ribosome
  • RNA: fMet-tRNAiMet
  • Protein or peptide: Initiation Factor 2
KeywordsInitiation Factor 2 / ribosome / translation / translation initiation / IF2 / translational GTPase / GTPase
Function / homology
Function and homology information


guanosine tetraphosphate binding / stringent response / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / ribosomal small subunit binding / RNA folding / transcriptional attenuation / positive regulation of ribosome biogenesis ...guanosine tetraphosphate binding / stringent response / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / ribosomal small subunit binding / RNA folding / transcriptional attenuation / positive regulation of ribosome biogenesis / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / translational termination / negative regulation of cytoplasmic translation / four-way junction DNA binding / DnaA-L2 complex / translation repressor activity / : / negative regulation of translational initiation / regulation of mRNA stability / negative regulation of DNA-templated DNA replication initiation / translation initiation factor activity / mRNA regulatory element binding translation repressor activity / assembly of large subunit precursor of preribosome / response to cold / positive regulation of RNA splicing / ribosome assembly / transcription elongation factor complex / regulation of DNA-templated transcription elongation / cytosolic ribosome assembly / response to reactive oxygen species / DNA endonuclease activity / transcription antitermination / translational initiation / regulation of cell growth / DNA-templated transcription termination / response to radiation / maintenance of translational fidelity / mRNA 5'-UTR binding / ribosome biogenesis / regulation of translation / large ribosomal subunit / transferase activity / ribosome binding / ribosomal small subunit biogenesis / ribosomal small subunit assembly / small ribosomal subunit / 5S rRNA binding / ribosomal large subunit assembly / cytosolic small ribosomal subunit / large ribosomal subunit rRNA binding / small ribosomal subunit rRNA binding / cytosolic large ribosomal subunit / cytoplasmic translation / tRNA binding / negative regulation of translation / rRNA binding / ribosome / structural constituent of ribosome / translation / response to antibiotic / negative regulation of DNA-templated transcription / GTPase activity / mRNA binding / 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 / : ...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 / : / Elongation factor G domain 2 / Translation initiation factor IF- 2 / Translation initiation factor IF-2, domain 3 superfamily / Putative DNA-binding domain superfamily / Translation elongation factor EFTu-like, domain 2 / Ribosomal protein S21, conserved site / Ribosomal protein S21 signature. / Ribosomal protein L11, bacterial-type / Elongation factor Tu domain 2 / Ribosomal protein L25, short-form / Ribosomal protein S14, bacterial/plastid / Ribosomal protein S21 superfamily / Ribosomal protein S16, conserved site / Ribosomal protein S16 signature. / Ribosomal protein S21 / 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 L11 signature. / : / Ribosomal protein L16 signature 1. / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / Ribosomal protein L16 signature 2. / Ribosomal protein L16, conserved site / Ribosomal protein L6, conserved site / Ribosomal protein L6 signature 1. / 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, N-terminal / Ribosomal protein L11, N-terminal domain / Ribosomal protein L17 signature. / 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, RNA binding domain / Ribosomal L25p family / Ribosomal protein L25 / Ribosomal protein L36 signature. / Ribosomal protein L32p, bacterial type / Ribosomal protein L28/L24 superfamily / Ribosomal protein L25/Gln-tRNA synthetase, N-terminal / 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 L33, conserved site / Ribosomal protein L33 signature. / Ribosomal protein L35, conserved site / Ribosomal protein L35 signature. / Ribosomal protein L28 / Ribosomal protein L35, non-mitochondrial / : / Ribosomal protein L5, bacterial-type / Ribosomal protein L18, bacterial-type / : / Ribosomal protein S19, bacterial-type / Ribosomal protein S3, bacterial-type / Ribosomal protein L6, bacterial-type / Ribosomal protein S13, bacterial-type / Ribosomal protein L9/RNase H1, N-terminal / Ribosomal protein S6, conserved site / Ribosomal protein S6 signature. / Ribosomal protein S7, bacterial/organellar-type / Ribosomal protein S9, bacterial/plastid / Ribosomal protein S11, bacterial-type / Ribosomal protein S20 / Ribosomal protein S20 superfamily / Ribosomal protein S20 / Ribosomal protein L36 / Ribosomal protein L36 superfamily / Ribosomal protein L36 / Ribosomal protein S4, bacterial-type / 30S ribosomal protein S17 / Ribosomal protein S5, bacterial-type / Ribosomal protein L19, conserved site / Ribosomal protein L19 signature. / Ribosomal protein L27, conserved site / Ribosomal protein L27 signature. / Ribosomal protein L20 signature. / Ribosomal protein L22, bacterial/chloroplast-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 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 ...Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL15 / Translation initiation factor IF-2 / 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: 4.6 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: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.8
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 1.8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jcn
  • Surface level: 1.8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jcn
  • Surface level: 1.8
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3285.png

Downloads & links

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Map

FileDownload / File: emd_3285.map.gz / Format: CCP4 / Size: 100.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationInitiation Factor 2 was stalled on Escherichia coli 70S ribosomes by the non-hydrolysable GTP analogue GDPNP in the presence of fMet-tRNAiMet and mRNA.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.23 Å/pix.
x 300 pix.
= 369. Å		1.23 Å/pix.
x 300 pix.
= 369. Å		1.23 Å/pix.
x 300 pix.
= 369. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.23 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.8 / Movie #1: 1.8
Minimum - Maximum-4.18712282 - 10.247615809999999
Average (Standard dev.)0.07194872 (±0.5593707)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 369.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.231.231.23
M x/y/z300300300
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/NS300300300
D min/max/mean-4.18710.2480.072

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Supplemental data

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Sample components

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Entire : Initiation Factor 2 was stalled on Escherichia coli 70S ribosomes...

EntireName: 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.
Components
  • Sample: 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.
  • Complex: 70S ribosome
  • RNA: fMet-tRNAiMet
  • Protein or peptide: Initiation Factor 2

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Supramolecule #1000: Initiation Factor 2 was stalled on Escherichia coli 70S ribosomes...

SupramoleculeName: 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
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
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
Source (natural)Organism: Escherichia coli (E. coli) / synonym: 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
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
Source (natural)Organism: Escherichia coli (E. coli) / synonym: 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: 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
Source (natural)Organism: Escherichia coli (E. coli) / synonym: 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 -mercapthoethanol, 2 mM spermidine and 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

Microscopy ID1
MicroscopeFEI POLARA 300
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 39000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / 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
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Electron microscopy #2

Microscopy ID2
MicroscopeFEI POLARA 300
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 39000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / 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
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsTo avoid overfitting, the data was refined in a resolution-limited scheme using SPIDER
CTF correctionDetails: CTFFIND4
Final reconstructionApplied 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

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