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- PDB-3iz4: Modified E. coli tmRNA in the resume state with the tRNA-like dom... -

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Basic information

Entry
Database: PDB / ID: 3iz4
TitleModified E. coli tmRNA in the resume state with the tRNA-like domain in the ribosomal P site interacting with the SmpB
Components
  • Modified E. coli transfer-messenger RNA
  • SsrA-binding protein
KeywordsRNA BINDING PROTEIN/RNA / transfer-messenger RNA / trans-translation / RNA / molecular mimicry / pseudo-knots / tRNA-like domain / mRNA-like domain / MS2 / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


trans-translation / rRNA binding / cytoplasm
Similarity search - Function
SsrA-binding protein / SsrA-binding protein, conserved site / Small protein B / SmpB protein / SsrA-binding protein.
Similarity search - Domain/homology
RNA / RNA (> 10) / RNA (> 100) / SsrA-binding protein
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 13.6 Å
AuthorsHashem, Y. / Fu, J. / Frank, J.
CitationJournal: EMBO J / Year: 2010
Title: Visualizing the transfer-messenger RNA as the ribosome resumes translation.
Authors: Jie Fu / Yaser Hashem / Iwona Wower / Jianlin Lei / Hstau Y Liao / Christian Zwieb / Jacek Wower / Joachim Frank /
Abstract: Bacterial ribosomes stalled by truncated mRNAs are rescued by transfer-messenger RNA (tmRNA), a dual-function molecule that contains a tRNA-like domain (TLD) and an internal open reading frame (ORF). ...Bacterial ribosomes stalled by truncated mRNAs are rescued by transfer-messenger RNA (tmRNA), a dual-function molecule that contains a tRNA-like domain (TLD) and an internal open reading frame (ORF). Occupying the empty A site with its TLD, the tmRNA enters the ribosome with the help of elongation factor Tu and a protein factor called small protein B (SmpB), and switches the translation to its own ORF. In this study, using cryo-electron microscopy, we obtained the first structure of an in vivo-formed complex containing ribosome and the tmRNA at the point where the TLD is accommodated into the ribosomal P site. We show that tmRNA maintains a stable 'arc and fork' structure on the ribosome when its TLD moves to the ribosomal P site and translation resumes on its ORF. Based on the density map, we built an atomic model, which suggests that SmpB interacts with the five nucleotides immediately upstream of the resume codon, thereby determining the correct selection of the reading frame on the ORF of tmRNA.
History
DepositionSep 21, 2010Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 20, 2010Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Jul 18, 2018Group: Data collection / Category: em_image_scans / em_software / Item: _em_software.image_processing_id
Revision 1.3Feb 21, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: Modified E. coli transfer-messenger RNA
B: SsrA-binding protein


Theoretical massNumber of molelcules
Total (without water)135,7422
Polymers135,7422
Non-polymers00
Water0
1


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

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Components

#1: RNA chain Modified E. coli transfer-messenger RNA


Mass: 121695.953 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: MS2-tmRNA (H8), a variant of E. coli tmRNAH8 that can bind the coat protein from MS2 bacteriophage, was constructed by PCR-directed mutagenesis.
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli)
#2: Protein SsrA-binding protein


Mass: 14046.285 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli) / References: UniProt: Q8RR57
Sequence detailsTHE MODEL IS BASED ON THE CRYO-EM MAP OBTAINED FROM E. COLI. THE CHAIN B OF THE MODEL CONTAINS THE ...THE MODEL IS BASED ON THE CRYO-EM MAP OBTAINED FROM E. COLI. THE CHAIN B OF THE MODEL CONTAINS THE SEQUENCE FROM T. THERMOPHILUS.

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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

ComponentName: Modified transfer-messenger RNA bound to the ribosomal P site
Type: RIBOSOME
Buffer solutionName: Polymix buffer / pH: 7.5 / Details: Polymix buffer
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE
Details: Vitrification done with vitrobot plunging into liquid ethane.

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 59000 X / Calibrated magnification: 100000 X / Nominal defocus max: 4500 nm / Nominal defocus min: 2000 nm
Specimen holderSpecimen holder model: OTHER / Specimen holder type: CARTRIDGE
Image recordingElectron dose: 24 e/Å2 / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k)
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1NAMDmodel fitting
2VMDmodel fitting
3SPIDER3D reconstruction
CTF correctionDetails: Volume
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionMethod: reference-based projection / Resolution: 13.6 Å / Num. of particles: 20873 / Nominal pixel size: 3 Å / Actual pixel size: 3 Å / Magnification calibration: 100000 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: METHOD--molecular dynamic flexible fitting REFINEMENT PROTOCOL--molecular dynamic flexible fitting
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms965 8053 0 0 9018

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