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Title:Cryo-EM reveals an active role for aminoacyl-tRNA in the accommodation process.
Authors:Valle M, Sengupta J, Swami NK, Grassucci RA, Burkhardt N, Nierhaus KH, Agrawal RK, Frank J
Sample:70S aa-tRNA-EF-Tu.GDP.kirromycin complex from E.coli
Aggregation state:Single particle (16.8 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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bullet_white.gif  Visualization
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bullet_white.gif  Map information
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Sample
Sample name: 70S aa-tRNA-EF-Tu.GDP.kirromycin complex from E.coli
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 ribosome10 pmol
2ligandaa-tRNAfalse14 pmol; fMet-tRNA(fMet) at the P site and Phe-tRNA(phe) at the A site
3ligandEF-Tufalse20 pmol
4ligandGDPtrue8 ul of 2.5mM; [Alpha-(32)P]GTP The radioactivity contributed by the Alpha-(32)P moiety of the GDP served to calculate a ~50% occupancy for the elongation factor.
5ligandkirromycinfalse3 ul of 100 mM; antibiotic
6ligandMF-mRNAtrue15 pmol; M(Met).F(Phe)-mRNA
Experiment
Sample preparation:
pHSample conc.DetailsStainingSample support details
7.5 mg/mL20 mM HEPES-KOH, 6mM MgCl2, 150mM NH4Cl, 4mM 2-metcaptoethanol, 0.05 mM spermine and 2 mM spermidine
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE30%93 Ktwo side blotting plungerBlot for 2 seconds before plunging ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI/PHILIPS EM420100 kVFLOOD BEAMBRIGHT FIELD2.0 mm1041 nm3844 nm4900052000LAB6Kodak SO163 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 mrad10 e/Å2Microscope: Philips EM420 Imaging date: 19971998-06-01
Processing
Software:SPIDER
CTF correction:defocus groups
Resolution by author:16.8 Å
Resolution method:FSC at 0.5 cut-off
Processing details:Final map was calculated from 8 CTF corrected defocus groups
Unit cell:
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
4514 μm/pixel1.212linkZEISS SCAI
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails
1EFC Rigid Bodycross correlation coefficientmanualThe EF-Tu domains were separately fitted by manual docking using program O (pdb codes for fitted coordinates: 1LS2, 1LU3)
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