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- EMDB-5784: EttA-bound E. coli 70S ribosome complex containing P-site tRNA an... -

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Basic information

Entry
Database: EMDB / ID: EMD-5784
TitleEttA-bound E. coli 70S ribosome complex containing P-site tRNA and A-site tRNA
Map dataReconstruction of E. coli 70S ribosome complex containing A-site tRNAPhe, P-site tRNAfMet, and EttA-EQ2 mutant protein
Sample
  • Sample: E. coli 70S ribosome complex 70S-EttA_EQ2-tRNAfMet-tRNAPhe
  • Complex: 70S ribosomeRibosome
  • Protein or peptide: Energy-dependent Translational Throttle A (EttA)
  • RNA: tRNAfMet
  • RNA: tRNAPhe
Keywordsprotein translation regulation / ABC-F protein family / ribosome / cryo-EM / single-molecule FRET / YjjK
Function / homology
Function and homology information


negative regulation of translational elongation / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / negative regulation of translational initiation / ribosomal small subunit assembly / cytosolic small ribosomal subunit / ribosome binding / ribosomal large subunit assembly / 5S rRNA binding / cytosolic large ribosomal subunit / cytoplasmic translation ...negative regulation of translational elongation / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / negative regulation of translational initiation / ribosomal small subunit assembly / cytosolic small ribosomal subunit / ribosome binding / ribosomal large subunit assembly / 5S rRNA binding / cytosolic large ribosomal subunit / cytoplasmic translation / tRNA binding / negative regulation of translation / ribosome / rRNA binding / structural constituent of ribosome / translation / response to antibiotic / mRNA binding / ATP hydrolysis activity / RNA binding / ATP binding / membrane / cytosol / cytoplasm
Similarity search - Function
Energy-dependent translational throttle protein EttA / ABC-transporter extension domain / ABC transporter / Ribosomal protein L1, bacterial-type / Ribosomal protein L1, conserved site / Ribosomal protein L1 / Ribosomal protein L1 signature. / Ribosomal protein L1, 3-layer alpha/beta-sandwich / Ribosomal protein L1-like / Ribosomal protein L1/ribosomal biogenesis protein ...Energy-dependent translational throttle protein EttA / ABC-transporter extension domain / ABC transporter / Ribosomal protein L1, bacterial-type / Ribosomal protein L1, conserved site / Ribosomal protein L1 / Ribosomal protein L1 signature. / Ribosomal protein L1, 3-layer alpha/beta-sandwich / Ribosomal protein L1-like / Ribosomal protein L1/ribosomal biogenesis protein / Ribosomal protein L1p/L10e family / ABC transporter-like, conserved site / ABC transporters family signature. / Ribosomal protein L33, conserved site / Ribosomal protein L33 signature. / Ribosomal protein L5, bacterial-type / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / Ribosomal protein S7, bacterial/organellar-type / Ribosomal protein L33 / Ribosomal protein L33 / Ribosomal protein L33 superfamily / Ribosomal protein L5, conserved site / Ribosomal protein L5 signature. / Ribosomal protein S7, conserved site / Ribosomal protein S7 signature. / Ribosomal protein L5, N-terminal / Ribosomal protein L5 / Ribosomal protein L5, C-terminal / ribosomal L5P family C-terminus / Ribosomal protein L5 / Ribosomal protein L5 domain superfamily / Ribosomal protein S5/S7 / Ribosomal protein S7 domain / Ribosomal protein S7 domain superfamily / Ribosomal protein S7p/S5e / Zinc-binding ribosomal protein / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL1 / Large ribosomal subunit protein bL33 / Energy-dependent translational throttle protein EttA / Large ribosomal subunit protein uL5
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.5 Å
AuthorsChen B / Boel G / Hashem Y / Ning W / Fei J / Wang C / Gonzalez RL / Hunt JF / Frank J
CitationJournal: Nat Struct Mol Biol / Year: 2014
Title: The ABC-F protein EttA gates ribosome entry into the translation elongation cycle.
Authors: Grégory Boël / Paul C Smith / Wei Ning / Michael T Englander / Bo Chen / Yaser Hashem / Anthony J Testa / Jeffrey J Fischer / Hans-Joachim Wieden / Joachim Frank / Ruben L Gonzalez / John F Hunt /
Abstract: ABC-F proteins have evaded functional characterization even though they compose one of the most widely distributed branches of the ATP-binding cassette (ABC) superfamily. Herein, we demonstrate that ...ABC-F proteins have evaded functional characterization even though they compose one of the most widely distributed branches of the ATP-binding cassette (ABC) superfamily. Herein, we demonstrate that YjjK, the most prevalent eubacterial ABC-F protein, gates ribosome entry into the translation elongation cycle through a nucleotide-dependent interaction sensitive to ATP/ADP ratio. Accordingly, we rename this protein energy-dependent translational throttle A (EttA). We determined the crystal structure of Escherichia coli EttA and used it to design mutants for biochemical studies including enzymological assays of the initial steps of protein synthesis. These studies suggest that EttA may regulate protein synthesis in energy-depleted cells, which have a low ATP/ADP ratio. Consistently with this inference, EttA-deleted cells exhibit a severe fitness defect in long-term stationary phase. These studies demonstrate that an ABC-F protein regulates protein synthesis via a new mechanism sensitive to cellular energy status.
History
DepositionNov 8, 2013-
Header (metadata) releaseDec 25, 2013-
Map releaseJan 1, 2014-
UpdateFeb 19, 2014-
Current statusFeb 19, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 80
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 80
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j5s
  • Surface level: 80
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3j5s
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_5784.map.gz / Format: CCP4 / Size: 9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of E. coli 70S ribosome complex containing A-site tRNAPhe, P-site tRNAfMet, and EttA-EQ2 mutant protein
Voxel sizeX=Y=Z: 2.7116 Å
Density
Contour LevelBy AUTHOR: 80.0 / Movie #1: 80
Minimum - Maximum-150.118362429999991 - 338.728607179999983
Average (Standard dev.)3.62113333 (±39.947555540000003)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-67-67-66
Dimensions134134134
Spacing134134134
CellA=B=C: 363.3544 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.71159701492542.71159701492542.7115970149254
M x/y/z134134134
origin x/y/z0.0000.0000.000
length x/y/z363.354363.354363.354
α/β/γ90.00090.00090.000
start NX/NY/NZ-95-75153
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS-67-67-66
NC/NR/NS134134134
D min/max/mean-150.118338.7293.621

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Supplemental data

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Sample components

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Entire : E. coli 70S ribosome complex 70S-EttA_EQ2-tRNAfMet-tRNAPhe

EntireName: E. coli 70S ribosome complex 70S-EttA_EQ2-tRNAfMet-tRNAPhe
Components
  • Sample: E. coli 70S ribosome complex 70S-EttA_EQ2-tRNAfMet-tRNAPhe
  • Complex: 70S ribosomeRibosome
  • Protein or peptide: Energy-dependent Translational Throttle A (EttA)
  • RNA: tRNAfMet
  • RNA: tRNAPhe

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Supramolecule #1000: E. coli 70S ribosome complex 70S-EttA_EQ2-tRNAfMet-tRNAPhe

SupramoleculeName: E. coli 70S ribosome complex 70S-EttA_EQ2-tRNAfMet-tRNAPhe
type: sample / ID: 1000 / Number unique components: 4

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Supramolecule #1: 70S ribosome

SupramoleculeName: 70S ribosome / type: complex / ID: 1 / Recombinant expression: No / Database: NCBI / Ribosome-details: ribosome-prokaryote: ALL
Ref GOdivclassse qspanoncli ckpopupspa nclassgree n(this)spandata popltspanc lassquotlo adingbarqu otgtltimgs rcquotimgl oadinggifq uotdecodin gquotasync quotgtltsp angtdataur lajaxphp?m odetaxoamp ...
divclassse qspanoncli ckpopupspa nclassgree n(this)spandata popltspanc lassquotlo adingbarqu otgtltimgs rcquotimgl oadinggifq uotdecodin gquotasync quotgtltsp angtdataur lajaxphp?m odetaxoamp kGO3A00422 55ampajax1 classpoptr giGO004225 5ispandiv
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MRE600
Molecular weightExperimental: 2.7 MDa

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Macromolecule #1: Energy-dependent Translational Throttle A (EttA)

MacromoleculeName: Energy-dependent Translational Throttle A (EttA) / type: protein_or_peptide / ID: 1 / Name.synonym: YjjK / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K-12 MG1655
Molecular weightTheoretical: 60 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: K-12 MG1655 / Recombinant plasmid: pBAD
SequenceUniProtKB: Energy-dependent translational throttle protein EttA

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Macromolecule #2: tRNAfMet

MacromoleculeName: tRNAfMet / type: rna / ID: 2 / Classification: TRANSFER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #3: tRNAPhe

MacromoleculeName: tRNAPhe / type: rna / ID: 3 / Classification: TRANSFER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Escherichia coli (E. coli)

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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

Concentration1.2 mg/mL
BufferpH: 6.9
Details: 50 mM Tris acetate, 100 mM KCl, 5 mM NH4OAc, 3.5 mM Mg(OAc)2, 0.5 mM Ca(OAc)2, 0.1 mM EDTA, 1 mM spermidine, 5 mM putrescine, 6 mM 2-mercaptoethanol, 0.5 mM Mg-ATP
GridDetails: Quantifoil R2/4 300 mesh Cu EM grid, coated with thin carbon film, glow discharged in H2/O2
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 80 K / Instrument: FEI VITROBOT MARK IV
Method: Wait time 30 sec, blot time 8 sec, at 4 degrees Celsius

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Electron microscopy #1

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 110637 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 80000
Sample stageSpecimen holder: Single tilt cryoholder, liquid Nitrogen cooled
Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 80 K
Microscopy ID1
DetailsLow dose
DateApr 5, 2011
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 574 / Average electron dose: 17 e/Å2
Details: Used the automatic image collection program Leginon
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Electron microscopy #2

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 110637 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 80000
Sample stageSpecimen holder: Single tilt cryoholder, liquid Nitrogen cooled
Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 80 K
Microscopy ID2
DetailsLow dose
DateJun 6, 2011
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 1816 / Average electron dose: 17 e/Å2
Details: Used the automatic image collection program Leginon
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each micrograph
Final reconstructionResolution.type: BY AUTHOR / Resolution: 7.5 Å / Resolution method: OTHER / Software - Name: SPIDER, RELION / Details: Subset after RELION 3D classification / Number images used: 39316
DetailsThe particles were selected via automatic particle picking followed by visual verification. 3D classification and refinement were performed using RELION.

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Atomic model buiding 1

Initial modelPDB ID:

3r8o
PDB Unreleased entry

SoftwareName: NAMD
DetailsProtocol: Molecular dynamics flexible fitting (Trabuco et al. Flexible Fitting of Atomic Structures into Electron Microscopy Maps Using Molecular Dynamics. Structure (2008) vol. 16 (5) pp. 673-683)
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation coefficient
Output model

PDB-3j5s:
EttA binds to ribosome exit site and regulates translation by restricting ribosome and tRNA dynamics

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Atomic model buiding 2

Initial modelPDB ID:

3r8t
PDB Unreleased entry

SoftwareName: NAMD
DetailsProtocol: Molecular dynamics flexible fitting
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation coefficient
Output model

PDB-3j5s:
EttA binds to ribosome exit site and regulates translation by restricting ribosome and tRNA dynamics

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Atomic model buiding 3

Initial modelPDB ID:

2wdg
PDB Unreleased entry


Chain - #0 - Chain ID: V / Chain - #1 - Chain ID: Y
SoftwareName: NAMD
DetailsProtocol: Molecular dynamics flexible fitting
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation coefficient
Output model

PDB-3j5s:
EttA binds to ribosome exit site and regulates translation by restricting ribosome and tRNA dynamics

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Atomic model buiding 4

Initial modelPDB ID:
SoftwareName: NAMD
DetailsMatched the apo-EttA model (derived from PDB ID 4FIN) to CFTR NBD1 (PDB ID 2PZE) to model the ATP-bound form of EttA. Protocol: Molecular dynamics flexible fitting
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation coefficient
Output model

PDB-3j5s:
EttA binds to ribosome exit site and regulates translation by restricting ribosome and tRNA dynamics

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