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Title:A cryo-electron microscopic study of ribosome-bound termination factor RF2.
Authors:Rawat UB, Zavialov AV, Sengupta J, Valle M, Grassucci RA, Linde J, Vestergaard B, Ehrenberg M, Frank J
Sample:E.coli 70s ribosome-RF2(mutant) complex
Aggregation state:Single particle (10.9 angstroms resolution)
Red flagLatest update:2011-05-26
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bullet_white.gif  Summary
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bullet.png Experimental details
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Sample
Sample name: E.coli 70s ribosome-RF2(mutant) complex
Theoretical molecular weight of the sample: 2.5
Components:
ID Type Name Exp. MW (MDa) Oligomeric details Mutant Organism GO identifier InterPro identifier Virus identifier Details
1ribosome-prokaryoteE.coli 70s ribosome
2ligandP-tRNAfalse
3ligandE-tRNAfalse
4ligandMFTI-mRNAfalseZavialov et al., A posttermination ribosomal complex is the guanine nucleotide exchange factor for peptide release factor RF3. Cell. 107,1-20 (2001).
5ligandRF2 (GAQ) mutanttrue
Experiment
Sample preparation:
pHSample conc.DetailsStainingSample support details
7.50.09 mg/mLPolymix buffer,containing at final concentration 5mM potassium phosphate, 5mM magnesium acetate, 5mM ammonium chloride, 95 mM potassium chloride, 0.5 mM calcium chloride, 8mM putrescine, 1mM spermidine and 1mM dithioerythritol.
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE58%36 KTwo side blotting plungerBlot and Plunge ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI TECNAI F20200 kVFLOOD BEAMBRIGHT FIELD2.0 mm2250 nm4050 nm5000049696FIELD EMISSION GUNKodac SO163 film mm

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
Cryo-transferGATAN LIQUID NITROGEN0°0° eV93 K K K mrad20 e/Å22001-08-02
Processing
Software:SPIDER/WEB
CTF correction:Wiener filtration of defocus groups
Resolution by author:10.9 Å
Resolution method:FSC at 0.5 cut-off
Processing details:Frank, J. (2000) Three-Dimensional Cryoelectron Microscopy of Ribosomes, Methods of Enzymology (Ch.18) 317, 276-291.
Unit cell:
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
4114 μm/pixel12linkZEISS SCAI
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails
1M16 1MVR Omanual fitting using O
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