4V7D

Structure of the Ribosome with Elongation Factor G Trapped in the Pre-Translocation State (pre-translocation 70S*tRNA*EF-G structure)


ELECTRON MICROSCOPY
Sample
Pre-translocation 70S ribosome with A/P*, P/E hybrid state tRNA and EF-G bound
Sample Components
70S ribosome
Transfer RNA
Messenger RNA
Elongation Factor G
Specimen Preparation
Sample Aggregation StatePARTICLE
Vitrification InstrumentFEI VITROBOT MARK II
Cryogen NameETHANE
Sample Vitrification DetailsFreshly glow-disharged grids were loaded into an FEI Mark II Vitrobot and equilibrated to 95% relative humidity at 22 degrees Celsius. 2 microliters o ...Freshly 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.
3D Reconstruction
Reconstruction MethodSINGLE PARTICLE
Number of Particles85115
Reported Resolution (Å)7.6
Resolution MethodFSC 0.143 CUT-OFF
Other DetailsRefinement included data to 12 Angstrom resolution to limit FSC bias. See primary citation Supplementary Information for details. (Single particle det ...Refinement included data to 12 Angstrom resolution to limit FSC bias. See primary citation Supplementary Information for details. (Single particle details: Refinement and 3D classification performed by Frealign) (Single particle--Applied symmetry: C1)
Refinement Type
Symmetry TypePOINT
Point SymmetryC1
Map-Model Fitting and Refinement
IdRefinement SpaceRefinement ProtocolRefinement TargetOverall B ValueFitting ProcedureDetails
1 (3R8S)REALRIGID BODY FITCross-correlationREFINEMENT PROTOCOL--rigid body
2 (1MMS)REALRIGID BODY FITCross-correlationREFINEMENT PROTOCOL--rigid body DETAILS--An E. col ...REFINEMENT PROTOCOL--rigid body DETAILS--An E. coli homology model was used to rebuild the L11 stalk.
3 (1DD3)REALRIGID BODY FITCross-correlationREFINEMENT PROTOCOL--rigid body DETAILS--An E. col ...REFINEMENT PROTOCOL--rigid body DETAILS--An E. coli homology model was used to rebuild L12.
4 (3U4M)REALRIGID BODY FITCross-correlationREFINEMENT PROTOCOL--rigid body DETAILS--An E. col ...REFINEMENT PROTOCOL--rigid body DETAILS--An E. coli homology model was used to rebuild L1.
5 (1ZAV)REALRIGID BODY FITCross-correlationREFINEMENT PROTOCOL--rigid body DETAILS--An E. col ...REFINEMENT PROTOCOL--rigid body DETAILS--An E. coli homology model was used to rebuild L10.
Data Acquisition
Detector TypeFEI FALCON I (4k x 4k)
Electron Dose (electrons/Å**2)30
Imaging Experiment1
Date of Experiment2012-11-02
Temperature (Kelvin)
Microscope ModelFEI TITAN KRIOS
Minimum Defocus (nm)1150
Maximum Defocus (nm)6950
Minimum Tilt Angle (degrees)
Maximum Tilt Angle (degrees)
Nominal CS0.01
Imaging ModeBRIGHT FIELD
Specimen Holder ModelFEI TITAN KRIOS AUTOGRID HOLDER
Nominal Magnification133333
Calibrated Magnification134615
SourceFIELD EMISSION GUN
Acceleration Voltage (kV)300
Imaging Details
EM Software
TaskSoftware PackageVersion
CTF CORRECTIONCTFFIND3
MODEL FITTINGCNS
MODEL FITTINGUCSF Chimera
RECONSTRUCTIONEMAN2
RECONSTRUCTIONFREALIGN
RECONSTRUCTIONIMAGIC
RECONSTRUCTIONRSAMPLE
Image Processing
CTF Correction TypeCTF Correction DetailsNumber of Particles SelectedParticle Selection Details
CTFFIND3, FREALIGN per micrograph