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- PDB-3j5s: EttA binds to ribosome exit site and regulates translation by res... -

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Basic information

Entry
Database: PDB / ID: 3j5s
TitleEttA binds to ribosome exit site and regulates translation by restricting ribosome and tRNA dynamics
Components
  • (50S ribosomal protein ...) x 3
  • 16S ribosomal RNA
  • 23S ribosomal RNA
  • 30S ribosomal protein S7
  • Energy-dependent translational throttle A (EttA)
  • P-site tRNA FMet
KeywordsRIBOSOME/TRANSLATION / protein translation regulation / ABC-F protein family / single-molecule FRET / YjjK / RIBOSOME-TRANSLATION complex
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 / ribosomal large subunit assembly / cytosolic small ribosomal subunit / ribosome binding / cytoplasmic translation / 5S rRNA binding / cytosolic large ribosomal subunit ...negative regulation of translational elongation / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / negative regulation of translational initiation / ribosomal small subunit assembly / ribosomal large subunit assembly / cytosolic small ribosomal subunit / ribosome binding / cytoplasmic translation / 5S rRNA binding / cytosolic large ribosomal subunit / tRNA binding / negative regulation of translation / rRNA binding / ribosome / 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 / Ribosomal protein S7, bacterial/organellar-type / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / Ribosomal protein L33 / Ribosomal protein L33 / Ribosomal protein L33 superfamily / Ribosomal protein L5, conserved site / Ribosomal protein S7, conserved site / Ribosomal protein L5, N-terminal / Ribosomal protein L5 signature. / Ribosomal protein L5, C-terminal / Ribosomal protein L5 / Ribosomal protein L5 domain superfamily / Ribosomal protein L5 / ribosomal L5P family C-terminus / Ribosomal protein S7 signature. / 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
: / : / RNA / RNA (> 10) / RNA (> 100) / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.5 Å
AuthorsHashem, Y.
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.
History
DepositionNov 15, 2013Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 8, 2014Provider: repository / Type: Initial release
Revision 1.1Jan 22, 2014Group: Database references
Revision 1.2Feb 19, 2014Group: Database references
Revision 1.3Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.image_processing_id / _em_software.name
Revision 1.4Feb 21, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _struct_ref_seq_dif.details

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
B: 16S ribosomal RNA
A: 23S ribosomal RNA
E: P-site tRNA FMet
D: Energy-dependent translational throttle A (EttA)
F: 50S ribosomal protein L1
G: 50S ribosomal protein L5
H: 50S ribosomal protein L33
I: 30S ribosomal protein S7


Theoretical massNumber of molelcules
Total (without water)304,6848
Polymers304,6848
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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RNA chain , 3 types, 3 molecules BAE

#1: RNA chain 16S ribosomal RNA /


Mass: 32636.531 Da / Num. of mol.: 1 / Fragment: SEE REMARK 999 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: GenBank: J01695.2
#2: RNA chain 23S ribosomal RNA /


Mass: 116242.516 Da / Num. of mol.: 1 / Fragment: SEE REMARK 999 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: GenBank: 33357927
#3: RNA chain P-site tRNA FMet


Mass: 24802.785 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli)

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Protein , 2 types, 2 molecules DI

#4: Protein Energy-dependent translational throttle A (EttA) / ABC transporter ATP-binding protein YjjK


Mass: 63357.668 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12 substr. MG1655 / Gene: yjjK / Plasmid: pBAD / Production host: Escherichia coli (E. coli) / Strain (production host): K12 substr. MG1655 / References: UniProt: P0A9W3
#8: Protein 30S ribosomal protein S7 /


Mass: 16861.523 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: P02359

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50S ribosomal protein ... , 3 types, 3 molecules FGH

#5: Protein 50S ribosomal protein L1 /


Mass: 24765.660 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: P0A7L0
#6: Protein 50S ribosomal protein L5 /


Mass: 20202.416 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: P62399
#7: Protein/peptide 50S ribosomal protein L33 /


Mass: 5814.842 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: P0A7N9

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Details

Sequence detailsTHE FULL RIBOSOME WAS IMAGED, BUT ONLY A SUBSET OF THE 16S AND 23S RIBOSOMAL RNA WAS MODELED IN THIS ENTRY.

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeParent-ID
1E. coli 70S ribosome complex 70S-(EttA-EQ2)-(fMet-tRNA)-(Phe-tRNA)RIBOSOME0
270S ribosomeRibosomeRIBOSOME1
3Energy-dependent Translational Throttle A (EttA)1
4fMet-tRNA1
5Phe-tRNA1
Buffer solutionName: 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
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
SpecimenConc.: 1.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Quantifoil 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 / Temp: 80 K / Humidity: 100 %
Details: Wait time 30 sec, blot time 8 sec (4 C), plunge into liquid ethane (FEI VITROBOT MARK IV)
Method: Wait time 30 sec, blot time 8 sec, at 4 degrees Celsius

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20 / Date: Apr 5, 2013
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 80000 X / Calibrated magnification: 110637 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1200 nm / Cs: 2 mm
Specimen holderSpecimen holder model: GATAN LIQUID NITROGEN
Specimen holder type: Single tilt cryoholder, liquid Nitrogen cooled
Temperature: 80 K
Image recordingElectron dose: 17 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Details: Low dose
Image scansNum. digital images: 1816
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1NAMDmodel fitting
2RELION3D reconstruction
3SPIDER3D reconstruction
CTF correctionDetails: Each micrograph
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 7.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 39316 / Nominal pixel size: 2.7116 Å / Actual pixel size: 2.7116 Å / Details: Subset after RELION 3D classification / Refinement type: HALF-MAPS REFINED INDEPENDENTLY / Symmetry type: POINT
Atomic model building
IDProtocolSpaceTarget criteriaDetails
1FLEXIBLE FITREALCross-correlation coefficientREFINEMENT PROTOCOL--FLEXIBLE DETAILS--Protocol- Molecular dynamics flexible fitting
2FLEXIBLE FITREALCross-correlation coefficientREFINEMENT PROTOCOL--FLEXIBLE DETAILS--Protocol- Molecular dynamics flexible fitting
3FLEXIBLE FITREALCross-correlation coefficientREFINEMENT PROTOCOL--FLEXIBLE DETAILS--Protocol- Molecular dynamics flexible fitting
4FLEXIBLE FITREALCross-correlation coefficientREFINEMENT PROTOCOL--FLEXIBLE DETAILS--Matched the apo-EttA model (derived from PDB ENTRY 4FIN) to CFTR NBD1 (PDB ENTRY 2PZE) to model the ATP-bound form of EttA. Protocol- Molecular dynamics flexible fitting
Atomic model building

Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-ID 3D fitting-IDAccession codeInitial refinement model-ID
13R8O

3r8o
PDB Unreleased entry

13R8O1
23R8T

3r8t
PDB Unreleased entry

23R8T2
32WDG

2wdg
PDB Unreleased entry

V32WDG3
42WDG

2wdg
PDB Unreleased entry

Y32WDG3
54FIN

4fin

44FIN4
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms9138 6058 0 0 15196

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