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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
DescriptorSsrA-binding protein/RNA Complex
KeywordsRNA BINDING PROTEIN/RNA / tmRNA / SmpB / RNA BINDING PROTEIN-RNA COMPLEX
Specimen sourceThermus thermophilus / bacteria / thermophilic / サームス・サーモフィラス
MethodElectron microscopy (13.6 A resolution / Single particle / Vitreous ice (cryo EM))
AuthorsFrank, J. / Felden, B. / Gillet, R. / Li, W.
CitationJ. Biol. Chem., 2007, 282, 6356-6363

primary. J. Biol. Chem., 2007, 282, 6356-6363 StrPapers
Scaffolding as an organizing principle in trans-translation. The roles of small protein B and ribosomal protein S1.
Reynald Gillet / Sukhjit Kaur / Wen Li / Marc Hallier / Brice Felden / Joachim Frank

#1. Proc.Natl.Acad.Sci.USA, 2006, 103, 16484-16489
Cryo-EM visualization of transfer messenger RNA with two SmpBs in a stalled ribosome
Kaur, S. / Gillet, R. / Li, W. / Gursky, R. / Frank, J.

#2. Science, 2003, 300, 127-130
Visualizing tmRNA entry into a stalled ribosome
Valle, M. / Gillet, R. / Kaur, K. / Henne, A. / Ramakrishnan, V. / Frank, J.

DateDeposition: Dec 18, 2006 / Release: Jan 23, 2007 / Last modification: Feb 24, 2009
Remark 999SEQUENCE 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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Structure visualization

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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)170,5994
Polyers170,5994
Non-polymers00
Water0
#1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: RNA chaintransfer-messenger RNA


Mass: 105729.453 Da / Num. of mol.: 1
Source: (natural) Thermus thermophilus / bacteria / thermophilic / サームス・サーモフィラス
#2: RNA chain16S ribosomal RNA


Mass: 27987.885 Da / Num. of mol.: 1 / Details: helix 44 of 30S ribosomal subunit
Source: (natural) Thermus thermophilus / bacteria / thermophilic / サームス・サーモフィラス
#3: Polypeptide(L)SsrA-binding protein


Mass: 18440.992 Da / Num. of mol.: 2
Source: (natural) Thermus thermophilus / bacteria / thermophilic / サームス・サーモフィラス

Cellular component

Molecular function

Biological process

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentReconstruction method: SINGLE PARTICLE / Specimen type: VITREOUS ICE (CRYO EM)

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Sample preparation

Assembly of specimenName: Binding of transfer messenger RNA with two SmpBs / Aggregation state: PARTICLE
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.]
ComponentName: Pre-accommodated ribosomal trans-translation complex: T. thermophilus 70S-mRNA-(P-site tRNA)-tmRNA-(SmpB)2-(EF-Tu)-GDP-kirromycin
Buffer solutionName: polimix
Sample preparationpH: 7.5 / Sample conc.: 32 mg/ml
Specimen supportDetails: This grid plus sample was kept at -80 degree C for several days before use.
VitrificationDetails: Blot for 5 seconds before plunging. Rapid plunge freezing in liquid ethane.

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Electron microscopy imaging

MicroscopyMicroscope model: F20 / Date: Jun 1, 2004
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 1500 e/A2 / Illumination mode: FLOOD BEAM LOW DOSE
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 deg. / Tilt angle min: 0 deg.
CameraType: KODAK SO163 FILM
RadiationDiffraction protocol: SINGLE WAVELENGTH / Monochromatic or laue m l: M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

Image selectionSoftware name: O
EM single particle entitySymmetry type: ASYMMETRIC
3D reconstructionMethod: single particle reconstruction / Resolution: 13.6 A / Nominal pixel size: 2.82 A/pix
CTF correction method: CTF correction of each defocus group reconstruction, by Wiener filtering
Atomic model buildingMethod: manual fitting using stereo visualization / Software name: O / Ref protocol: Manual / Ref space: REAL
Atomic model buildingPDB-ID: 1P6V for SmpB and TLD of tmRNA
Number of atoms included #LASTProtein: 237 / Nucleic acid: 414 / Ligand: 0 / Solvent: 0 / Total: 651

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