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- EMDB-5841: EttA-bound E. coli 70S ribosome complex containing P-site tRNA an... -

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Basic information

Entry
Database: EMDB / ID: EMD-5841
TitleEttA-bound E. coli 70S ribosome complex containing P-site tRNA and A-site tRNA (raw map)
Map dataReconstruction of E. coli 70S ribosome complex containing A-site tRNAPhe, P-site tRNAfMet, and EttA-EQ2 mutant protein. This entry is the raw map of EMD-5784.
Sample
  • Sample: E. coli 70S ribosome complex 70S-EttA_EQ2-tRNAfMet-tRNAPhe
  • Complex: 70S ribosome
  • 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 / ribosome binding / tRNA binding / rRNA binding / translation / ATP hydrolysis activity / ATP binding / cytoplasm
Similarity search - Function
Energy-dependent translational throttle protein EttA / ABC-transporter extension domain / ABC transporter / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Energy-dependent translational throttle protein EttA
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
DepositionDec 16, 2013-
Header (metadata) releaseJan 1, 2014-
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: 0.07
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5841.png

Downloads & links

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Map

FileDownload / File: emd_5841.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. This entry is the raw map of EMD-5784.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.71 Å/pix.
x 134 pix.
= 363.354 Å		2.71 Å/pix.
x 134 pix.
= 363.354 Å		2.71 Å/pix.
x 134 pix.
= 363.354 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.7116 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07 / Movie #1: 0.07
Minimum - Maximum-0.08620983 - 0.25052011
Average (Standard dev.)0.00436634 (±0.03434465)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
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/NS000
NC/NR/NS134134134
D min/max/mean-0.0860.2510.004

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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 ribosome
  • 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 GO0: GO:0042255
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

Microscopy ID1
MicroscopeFEI TECNAI F20
TemperatureAverage: 80 K
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
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 110637 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / 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
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Electron microscopy #2

Microscopy ID2
MicroscopeFEI TECNAI F20
TemperatureAverage: 80 K
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
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 110637 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / 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
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsThe particles were selected via automatic particle picking followed by visual verification. 3D classification and refinement were performed using RELION.
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. This map is the raw map for entry EMD-5784. EMD-5784 has been amplitude-corrected and low-pass filtered to the reported resolution.
Number images used: 39316

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