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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:Pre-translocation ribosome with bound A/P, P/E hybrid state tRNA
Method:Single particle reconstruction (7.6 angstroms resolution)
Related entries in frame:EMD-5796, EMD-5797, EMD-5798, EMD-5800
Red flagLatest update:2014-07-23
Sample
Sample name: Pre-translocation ribosome with bound A/P, P/E hybrid state 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
2nucleic-acidTransfer RNA0.025falseEscherichia coli
3nucleic-acidMessenger RNA0.012truesynthetic construct
4ligandViomycin0.001falseunidentified
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:85115
Imposed symmetry:C1
Resolution by author:7.6 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
4GD1 rigid bodyCross-correlationChimera, CNSREALChain Y not used in refinement. See primary citation Supplementary Information for details.
3R8S rigid bodyCross-correlationChimera, CNSREALSee primary citation Supplementary Information for details.
1MMS rigid bodyCross-correlationChimera, CNSREALUsed to rebuild the L11 stalk.
3U4M rigid bodyCross-correlationChimera, CNSREALUsed to build homology model for L1.
1ZAV rigid bodyCross-correlationChimera, CNSREALUsed to build homology model for L10.