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Title:Structure of the Ribosome with Elongation Factor G Trapped in the Pre-Translocation State
Authors:Brilot AF, Korostelev AA, Ermolenko DN, Grigorieff N
Sample:Ribosome bound to EF-G and P/E tRNA
Method:Single particle reconstruction (7.3 angstroms resolution)
Related entries in frame:EMD-5796, EMD-5797, EMD-5799, EMD-5800
Red flagLatest update:2014-01-15
Sample
Sample name: Ribosome bound to EF-G and P/E tRNA
Theoretical molecular weight of the sample: 3
Components:
ID Type Name Exp. MW (MDa) Theo. MW (MDa) Oligomeric details Recombinant expression Synthetic Organism UniProt identifier GO identifier InterPro identifier Virus identifier Details
1ribosome-prokaryote70S ribosome3falseEscherichia coli
2proteinElongation Factor G0.078monomertrueEscherichia coliP0A6M8GO:0006414, GO:0005525, GO:0003746, GO:0005622IPR004540
3nucleic-acidTransfer RNA0.025falseEscherichia coli
4nucleic-acidMessenger RNA0.012truesynthetic construct
5ligandFusidic Acid0.001falseunidentified
6ligandViomycin0.001falseunidentified
Experiment
Specimen state: Particle
Specimen preparation:
pHSpecimen conc.DetailsStainingSpecimen support details
7.60.4 mg/mL10 mM HEPES-KOH, 5 mM MgCl2, 90 mM NH4Cl, 2 mM spermidine, 0.1 mM spermine, 6 mM BME, 0.5 mM viomycin, 0.5 mM GTP, 0.5 mM fusidic acidC-flat 1.2/1.3 holey carbon 400 mesh copper grid, glow discharged with a current of -20 mA for 45 seconds in an EMITECH K100X glow discharge unit
Vitrification:
Cryogen nameHumidityTemp.Instr.MethodTime resolvedDetails
ETHANE95% KFEI VITROBOT MARK IIFreshly glow-disharged grids were loaded into an FEI Mark II Vitrobot and equilibrated to 95% relative humidity at 22 degrees Celsius. 2 microliters of sample was applied through the side port, blotted for 7 seconds with a positional offset of 2, and plunged into liquid ethane. ms
Imaging:
MicroscopeVoltageIllumination modeImaging modeCsDefocus min.Defocus max.Nominal mag.Calibrated mag.Electron sourceDetectorDetector distanceAstigmatism
FEI TITAN KRIOS300 kVFLOOD BEAMBRIGHT FIELD0.01 mm1150 nm6950 nm133333134615FIELD EMISSION GUNFEI FALCON I (4k x 4k) mmAutomatically corrected using FEI software

Specimen holderHolder modelTilt min.Tilt max.Energy filterEnergy windowTemp.Temp. min.Temp. max.Beam tiltElectron doseOther detailsDate
FEI TITAN KRIOS AUTOGRID HOLDER°° eV K K K mrad30 e/Å202-NOV-2012
Processing
Protocol:Projection Matching
Software:EMAN2, IMAGIC, FREALIGN, RSAMPLE, CTFFIND3
CTF correction:CTFFIND3, FREALIGN per micrograph
Number of particles:1341961
Imposed symmetry:C1
Resolution by author:7.3 Å
Resolution method:FSC 0.143, semi-independent
Processing details:Refinement included data to 12 Angstrom resolution to limit FSC bias. See primary citation Supplementary Information for details. Refinement and 3D classification performed by Frealign. See primary citation Supplementary Information for details.
Scanned images:
Num. imagesSampling sizeOD rangeQuant. bit numberOther detailsScanner
1334114.0 μm/pixel16
Fitting:
PDBProtocolTarget crit.SoftwareB valueFitting spacePDB chainDetails