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Title:Structure of eEF3 and the mechanism of transfer RNA release from the E-site.
Authors:R.Beckmann and G.Andersen
Sample:80S-RNC-eEF3-AMP-PNP complex from S. cerevisiae
Aggregation state:Single particle (9.9 angstroms resolution)
Red flagLatest update:2011-09-02
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bullet_white.gif  Summary
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bullet.png Experimental details
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bullet_white.gif  Visualization
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Sample
Sample name: 80S-RNC-eEF3-AMP-PNP complex from S. cerevisiae
Oligomeric state: One ribosome binds to one molecule of eEF3
Theoretical molecular weight of the sample: 4.3
Components:
ID Type Name Exp. MW (MDa) Oligomeric details Recombinant expression Synthetic Organism GO identifier InterPro identifier Virus identifier Details
1ribosome-eukaryoteprogrammed 80S ribosome4.2programmed 80S ribosome with a P-site tRNA
2proteinelongation factor 30.115MonomertrueSaccharomyces cerevisiaeeEF3 with a C-terminal His-Tag and a factor
Experiment
Sample preparation:
pHSample conc.DetailsStainingSample support details
7.50.168 mg/mL20 mM HEPES, pH 7.5, 10 mM Mg(OAc)2, 150 mM KOAc 1 mM DTT, 0.05% Nikkol, 125 mM Sucrose,0.01 mg ml-1 Cycloheximide, 0.5 mM AMP-PNP, 0.1 mM Neomycin, 0.3 % DesoxyBigChapsCryo-EM, no stainingQuantifoil grids 200 mesh R2/4
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE95% KFEI VITROBOTBlot for 10 seconds before plunging, use 2 layers of filter paper msVitrification instrument: Vitrobot
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI TECNAI F30300 kVSPOT SCANBRIGHT FIELD2.27 mm1300 nm3500 nm3900038900FIELD EMISSION GUNKodak SO163 film mmobjective lens astigmatism was corrected at 100,000 time

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
Polara Multispecimen HolderOTHER°° eV95 K95 K K mrad20 e/Å22005-01-13
Processing
Software:SPIDER
Resolution by author:9.9 Å
Resolution method:FSC at 0.5 cutoff
Unit cell:
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
1414.35 μm/pixel1.216linkPRIMESCAN
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails
rigid bodycross correlationO and SitusThe domains were separately fitted by manual docking using program O
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