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- EMDB-5785: EttA-bound E. coli 70S ribosome complex containing P-site tRNA -

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Basic information

Entry
Database: EMDB / ID: EMD-5785
TitleEttA-bound E. coli 70S ribosome complex containing P-site tRNA
Map data
SampleE. coli 70S ribosome complex 70S-EttA_EQ2-tRNAfMet
  • ribosome-prokaryote
  • Energy-dependent Translational Throttle A (EttA)
  • nucleic-acidNucleic acid
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 / ATPase / ATP binding / cytoplasm
AAA+ ATPase domain / ABC transporter-like / ABC transporter, conserved site / Energy-dependent translational throttle protein EttA / P-loop containing nucleoside triphosphate hydrolase / ABC-transporter extension domain
Energy-dependent translational throttle protein EttA
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.1 Å
AuthorsChen B / Boel G / Hashem Y / Ning W / Fei J / Wang C / Gonzalez RL / Hunt JF / Frank J
Citation
Journal: Nat Struct Mol Biol / Year: 2014
Title: EttA regulates translation by binding the ribosomal E site and restricting ribosome-tRNA dynamics.
Authors: Bo Chen / Grégory Boël / Yaser Hashem / Wei Ning / Jingyi Fei / Chi Wang / Ruben L Gonzalez / John F Hunt / Joachim Frank /
Abstract: Cells express many ribosome-interacting factors whose functions and molecular mechanisms remain unknown. Here, we elucidate the mechanism of a newly characterized regulatory translation factor, ...Cells express many ribosome-interacting factors whose functions and molecular mechanisms remain unknown. Here, we elucidate the mechanism of a newly characterized regulatory translation factor, energy-dependent translational throttle A (EttA), which is an Escherichia coli representative of the ATP-binding cassette F (ABC-F) protein family. Using cryo-EM, we demonstrate that the ATP-bound form of EttA binds to the ribosomal tRNA-exit site, where it forms bridging interactions between the ribosomal L1 stalk and the tRNA bound in the peptidyl-tRNA-binding site. Using single-molecule fluorescence resonance energy transfer, we show that the ATP-bound form of EttA restricts ribosome and tRNA dynamics required for protein synthesis. This work represents the first example, to our knowledge, in which the detailed molecular mechanism of any ABC-F family protein has been determined and establishes a framework for elucidating the mechanisms of other regulatory translation factors.
#1: Journal: 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.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionNov 9, 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
  • Download
  • Surface view colored by height
  • Surface level: 80
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_5785.map.gz / Format: CCP4 / Size: 9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
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
Contour LevelBy AUTHOR: 80.0 / Movie #1: 80
Minimum - Maximum-104.432769780000001 - 305.265014650000012
Average (Standard dev.)5.22117186 (±35.891967770000001)
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-104.433305.2655.221

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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

EntireName: E. coli 70S ribosome complex 70S-EttA_EQ2-tRNAfMet / Number of components: 3

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Component #1: ribosome-prokaryote, 70S ribosome

Ribosome-prokaryoteName: 70S ribosomeRibosome / Prokaryote: ALL / Recombinant expression: No
MassExperimental: 2.7 MDa
SourceSpecies: Escherichia coli (E. coli) / Strain: MRE600
External referencesGene Ontology: ribosome assembly

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Component #2: protein, Energy-dependent Translational Throttle A (EttA)

ProteinName: Energy-dependent Translational Throttle A (EttA) / a.k.a: YjjK / Recombinant expression: Yes
MassTheoretical: 60 kDa
SourceSpecies: Escherichia coli (E. coli) / Strain: K-12 MG1655
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pBAD / Strain: K-12 MG1655
External referencesUniProt: Energy-dependent translational throttle protein EttA

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Component #3: nucleic-acid, tRNAfMet

nucleic acidName: tRNAfMet / Class: T-RNA / Structure: DOUBLE HELIX / Synthetic: No
SourceSpecies: Escherichia coli (E. coli)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 1.2 mg/mL
Buffer solution: 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
pH: 6.9
Support filmQuantifoil R2/4 300 mesh Cu EM grid, coated with thin carbon film, glow discharged in H2/O2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 80 K / Humidity: 100 %
Method: Wait time 30 sec, blot time 8 sec, at 4 degrees Celsius

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Electron microscopy imaging #1

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F20 / Date: Apr 5, 2011 / Details: Low dose
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 17 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 80000 X (nominal), 110637 X (calibrated) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1200 - 3500 nm
Specimen HolderHolder: Single tilt cryoholder, liquid Nitrogen cooled / Model: GATAN LIQUID NITROGEN / Temperature: 80
CameraDetector: GATAN ULTRASCAN 4000 (4k x 4k)

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Electron microscopy imaging #2

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F20 / Date: Jun 6, 2011 / Details: Low dose
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 17 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 80000 X (nominal), 110637 X (calibrated) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1200 - 3500 nm
Specimen HolderHolder: Single tilt cryoholder, liquid Nitrogen cooled / Model: GATAN LIQUID NITROGEN / Temperature: 80
CameraDetector: GATAN ULTRASCAN 4000 (4k x 4k)

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Image acquisition

Image acquisition #1Number of digital images: 574
Details: Used the automatic image collection program Leginon
Image acquisition #2Number of digital images: 1816
Details: Used the automatic image collection program Leginon

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 16639
Details: The particles were selected via automatic particle picking followed by visual verification. 3D classification and refinement were performed using RELION.
3D reconstructionSoftware: SPIDER, RELION / CTF correction: Each micrograph / Details: Subset after RELION 3D classification / Resolution: 9.1 Å / Resolution method: FSC 0.143, gold-standard

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