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Title:Three-dimensional structures of translating ribosomes by Cryo-EM.
Authors:Gilbert RJ, Fucini P, Connell S, Fuller SD, Nierhaus KH, Robinson CV, Dobson CM, Stuart DI
Sample:E. coli ribosome translating GFP and tandem Ig domains - difference map
Aggregation state:Single particle (16 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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Sample
Sample name: E. coli ribosome translating GFP and tandem Ig domains - difference map
Oligomeric state: 2 tRNA molecules and the average of two nascent proteins
Components:
ID Type Name Exp. MW (MDa) Oligomeric details Mutant Organism GO identifier InterPro identifier Virus identifier Details
1nucleic-acidP site tRNAfalseE. coli
2nucleic-acidE site tRNAfalseE. coli
3proteinnascent proteinmonomertruen/aDensity for nascent protein is average of that for tandem Ig domains and GFP
Experiment
Sample preparation:
pHSample conc.DetailsStainingSample support details
5.0 mg/mL20 mM HEPES, 150 mM ammonium acetate, 6 mM magnesium acetate, 2 mM spermidine, 0.05 mM spermine and 4 mM 2-mercaptoethanol. Concentration of ribosomes expressed to A260 units.300 mesh copper grid with holey carbon film
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE%100 KStandard unmodified guillotine plunger ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI/PHILIPS CM200FEG200 kVOTHEROTHER2 mm nm nm50000FIELD EMISSION GUNKodak SO163 film mm

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
EucentricGATAN LIQUID NITROGEN°° eV100 K K K mrad e/Å22000-05-02
Processing
Software:SPIDER, IMAGIC, GAP, CNS, XPLOR
CTF correction:Each negative dataset
Resolution by author:16 Å
Resolution method:FSC at 0.5 cut-off
Processing details:See entries 1068, 1070, 1071 and 1072. Vector difference maps were calculated between reconstructions of each nascent protein-ribosome complex and the control inactive map. The difference densities for the tandem Ig domains and GFP nascent proteins were more similar to each other than either was to the single Ig domain nascent protein. Therefore these two difference maps were averaged together to produce a set of consensus difference densities representing the main differences between inactive ribosomes and ribosomes stalled during translation that contain a nascent polypeptide chain.
Unit cell:
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
8.322 μm/pixel58linkScanner model: UMAX PowerLook 3000OTHER
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails
Rigid bodyR-factor and correlation coefficientGAPREAL
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