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- PDB-2ob7: Structure of tmRNA-(SmpB)2 complex as inferred from cryo-EM -

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Basic information

Entry
Database: PDB / ID: 2ob7
TitleStructure of tmRNA-(SmpB)2 complex as inferred from cryo-EM
Components
  • 16S ribosomal RNA
  • SsrA-binding protein
  • transfer-messenger RNA
KeywordsRNA BINDING PROTEIN/RNA / tmRNA / SmpB / RNA BINDING PROTEIN-RNA COMPLEX
Function / homology
Function and homology information


trans-translation / RNA binding / cytosol
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 speciesThermus thermophilus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 13.6 Å
AuthorsFrank, J. / Felden, B. / Gillet, R. / Li, W.
Citation
Journal: J Biol Chem / Year: 2007
Title: Scaffolding as an organizing principle in trans-translation. The roles of small protein B and ribosomal protein S1.
Authors: Reynald Gillet / Sukhjit Kaur / Wen Li / Marc Hallier / Brice Felden / Joachim Frank /
Abstract: A eubacterial ribosome stalled on a defective mRNA can be released through a quality control mechanism referred to as trans-translation, which depends on the coordinating binding actions of transfer- ...A eubacterial ribosome stalled on a defective mRNA can be released through a quality control mechanism referred to as trans-translation, which depends on the coordinating binding actions of transfer-messenger RNA, small protein B, and ribosome protein S1. By means of cryo-electron microscopy, we obtained a map of the complex composed of a stalled ribosome and small protein B, which appears near the decoding center. This result suggests that, when lacking a codon, the A-site on the small subunit is a target for small protein B. To investigate the role of S1 played in trans-translation, we obtained a cryo-electron microscopic map, including a stalled ribosome, transfer-messenger RNA, and small protein Bs but in the absence of S1. In this complex, several connections between the 30 S subunit and transfer-messenger RNA that appear in the +S1 complex are no longer found. We propose the unifying concept of scaffolding for the roles of small protein B and S1 in binding of transfer-messenger RNA to the ribosome during trans-translation, and we infer a pathway of sequential binding events in the initial phase of trans-translation.
#1: Journal: Proc.Natl.Acad.Sci.USA / Year: 2006
Title: Cryo-EM visualization of transfer messenger RNA with two SmpBs in a stalled ribosome
Authors: Kaur, S. / Gillet, R. / Li, W. / Gursky, R. / Frank, J.
#2: Journal: Science / Year: 2003
Title: Visualizing tmRNA entry into a stalled ribosome
Authors: Valle, M. / Gillet, R. / Kaur, K. / Henne, A. / Ramakrishnan, V. / Frank, J.
History
DepositionDec 18, 2006Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 23, 2007Provider: repository / Type: Initial release
Revision 1.1May 1, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Jul 18, 2018Group: Data collection / Category: em_image_scans / em_software / Item: _em_software.image_processing_id
Revision 1.4Dec 27, 2023Group: 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
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
Remark 999SEQUENCE The deposited entry is a model to fit the cryo-EM map of tmRNA+SmpB(2) from Thermus ...SEQUENCE The deposited entry is a model to fit the cryo-EM map of tmRNA+SmpB(2) from Thermus thermophilus. The deposition includes 4 chains: chain A: tmRNA model chains B,C: SmpB (from 1P6V.pdb chain A, for SmpB from AQUIFEX AEOLICUS); chain D: helix 44 of 30S ribosomal subunit (from 1N34.pdb chain A:1406-1496. X-ray structure of 1N34 is from Thermus thermophilus). The E.coli model eschcolitm3D-model-72.pdb (http://www.ag.auburn.edu/mirror/tmRDB/rna/tmrna.html/) was used as a template to build the model by replacing several fragments which have x-ray crystal as alternatives, and by fitting all the structures into the cryo-EM maps. Nucleotide numbering in this model follows E.coli sequence. Considering the differences between tmRNA sequences from E.coli and T.thermophilus, a small number of nucleotides in the template model are not included in this model.

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Assembly

Deposited unit
A: transfer-messenger RNA
D: 16S ribosomal RNA
B: SsrA-binding protein
C: SsrA-binding protein


Theoretical massNumber of molelcules
Total (without water)170,5984
Polymers170,5984
Non-polymers00
Water00
1


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

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Components

#1: RNA chain transfer-messenger RNA / Coordinate model: P atoms only


Mass: 105728.523 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus (bacteria)
#2: RNA chain 16S ribosomal RNA / Coordinate model: P atoms only


Mass: 27987.715 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: helix 44 of 30S ribosomal subunit / Source: (natural) Thermus thermophilus (bacteria)
#3: Protein SsrA-binding protein / Coordinate model: Cα atoms only


Mass: 18440.818 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus (bacteria) / References: UniProt: O66640*PLUS

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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: Pre-accommodated ribosomal trans-translation complex: T. thermophilus 70S-mRNA-(P-site tRNA)-tmRNA-(SmpB)2-(EF-Tu)-GDP-kirromycin
Type: RIBOSOME
Details: see Experimental Procedures in Kaur et al. (PNAS). [Deposition refers to structure of tmRNA-(SmpB)2 complex derived by fitting of EM map from Kaur et al. Fitting was modified in Gillet et al.]
Buffer solutionName: polimix / pH: 7.5 / Details: polimix
SpecimenConc.: 32 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: This grid plus sample was kept at -80 degree C for several days before use.
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE
Details: Blot for 5 seconds before plunging. Rapid plunge freezing in liquid ethane.

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20 / Date: Jun 1, 2004
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 55000 X / Calibrated magnification: 49000 X / Nominal defocus max: 3635 nm / Nominal defocus min: 1475 nm / Cs: 2 mm
Specimen holderTemperature: 296 K / Tilt angle max: 0 ° / Tilt angle min: 0 °
Image recordingElectron dose: 15 e/Å2 / Film or detector model: KODAK SO-163 FILM

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Processing

EM software
IDNameCategory
1Omodel fitting
2SPIDER3D reconstruction
CTF correctionDetails: CTF correction of each defocus group reconstruction, by Wiener filtering
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionMethod: single particle reconstruction / Resolution: 13.6 Å / Num. of particles: 52829 / Nominal pixel size: 2.82 Å / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Details: METHOD--manual fitting using stereo visualization REFINEMENT PROTOCOL--Manual
Atomic model buildingPDB-ID: 1P6V

1p6v


Accession code: 1P6V / Details: 1P6V FOR SMPB AND TLD OF TMRNA / Source name: PDB / Type: experimental model
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms237 414 0 0 651

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