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Title:Visualizing tmRNA entry into a stalled ribosome.
Authors:Valle M, Gillet R, Kaur S, Henne A, Ramakrishnan V, Frank J
Sample:70S-Psite tRNA - mRNA - EF-Tu.GDP - alanylated tmRNA - SmpB - Kirromycin - S1
Method:Single particle reconstruction (13 angstroms resolution)
Red flagLatest update:2012-10-24
Sample
Sample name: 70S-Psite tRNA - mRNA - EF-Tu.GDP - alanylated tmRNA - SmpB - Kirromycin - S1
Components:
ID Type Name Exp. MW (MDa) Oligomeric details Recombinant expression Synthetic Organism UniProt identifier GO identifier InterPro identifier Virus identifier Details
1ribosome-prokaryote70S RibosomefalseThermus thermophilus HB8Thermus thermophilus
2nucleic-acidtRNAfalseThermus thermophilus HB8P site
3proteinEF-TufalseThermus thermophilus HB8
4ligandtmRNAfalseThermus thermophilus HB8
5proteinSmpBfalseThermus thermophilus HB8
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
7.5 mg/mL25 mM Hepes-KOH pH 7.5, 30 mM NH4Cl, 7mM MgCl2, 2 mM ATP, 6 mM PEP, 10g/ml PK
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE90%93 KHOMEMADE PLUNGERBlot for 2 seconds before plunging msVitrification instrument: two side blotting plunger
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI TECNAI F20200 kVFLOOD BEAMBRIGHT FIELD2.0 mm1450 nm3700 nm4900052000FIELD EMISSION GUNKODAK SO-163 FILM mm

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
Cryo transferGATAN LIQUID NITROGEN°° eV93 K K K mrad15 e/Å22002-8-1
Processing
Protocol:reference projection
Software:SPIDER
CTF correction:defocus groups
Number of particles:27644
Resolution by author:13 Å
Resolution method:FSC at 0.15 cut-off
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
4414 μm/pixel1.212ZEISS SCAI
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails
rigid bodycross correlation coefficientmanualProtocol: Rigid Body. The domains were separately fitted by manual docking using program O