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- EMDB-5362: New mRNA-tRNA translocation intermediates revealed by cryoEM: Cla... -

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Basic information

Entry
Database: EMDB / ID: 5362
TitleNew mRNA-tRNA translocation intermediates revealed by cryoEM: Class 6 (rotated 70S ribosome with A-site and P-site tRNAs)
Keywordsribosome / 70S / rotated / intermediates / translocation / tRNA
SampleTrp-tRNA-EFTu-GDP-kir-70S ribosome
SourceEscherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /
Map dataCryo-EM-revealed rotated 70S ribosome with A and P site tRNAs (class 6)
Methodsingle particle reconstruction, at 11.5 Å resolution
AuthorsAgirrezabala X / Liao HY / Schreiner E / Fu J / Ortiz-Meoz RF / Schulten K / Green R / Frank J
CitationProc. Natl. Acad. Sci. U.S.A., 2012, 109, 6094-6099

Proc. Natl. Acad. Sci. U.S.A., 2012, 109, 6094-6099 StrPapers
Structural characterization of mRNA-tRNA translocation intermediates.
Xabier Agirrezabala / Hstau Y Liao / Eduard Schreiner / Jie Fu / Rodrigo F Ortiz-Meoz / Klaus Schulten / Rachel Green / Joachim Frank

Validation ReportPDB-ID: 4v6r

SummaryFull reportAbout validation report
DateDeposition: Dec 2, 2011 / Header (metadata) release: Jan 26, 2012 / Map release: Feb 16, 2012 / Last update: Jul 23, 2014

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.15
  • Imaged by UCSF CHIMERA
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  • Surface view colored by height
  • Surface level: 0.15
  • Imaged by UCSF CHIMERA
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  • Surface view with fitted model
  • Atomic models: : PDB-4v6r
  • Surface level: 0.15
  • Imaged by UCSF CHIMERA
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Supplemental images

Downloads & links

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Map

Fileemd_5362.map.gz (map file in CCP4 format, 61037 KB)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
250 pix
1.5 Å/pix.
= 375. Å
250 pix
1.5 Å/pix.
= 375. Å
250 pix
1.5 Å/pix.
= 375. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider package.

Voxel sizeX=Y=Z: 1.5 Å
Density
Contour Level:0.1 (by author), 0.15 (movie #1):
Minimum - Maximum-0.49196333 - 1.57512212
Average (Standard dev.)0.02891555 (0.20386297)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions250250250
Origin-124-124-124
Limit125125125
Spacing250250250
CellA=B=C: 375 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.51.51.5
M x/y/z250250250
origin x/y/z0.0000.0000.000
length x/y/z375.000375.000375.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-62-62-62
NX/NY/NZ125125125
MAP C/R/S123
start NC/NR/NS-124-124-124
NC/NR/NS250250250
D min/max/mean-0.4921.5750.029

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Supplemental data

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Sample components

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Entire Trp-tRNA-EFTu-GDP-kir-70S ribosome

EntireName: Trp-tRNA-EFTu-GDP-kir-70S ribosome / Number of components: 3 / Oligomeric State: monomeric
MassTheoretical: 2.8 MDa / Experimental: 2.8 MDa

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Component #1: ribosome-prokaryote, 70S ribosome

Ribosome-prokaryoteName: 70S ribosome / Prokaryote: ALL / Recombinant expression: No
SourceSpecies: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /

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Experimental details

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Sample preparation

Specimen stateparticle
Sample solutionSpecimen conc.: 0.03 mg/ml
Buffer solution: HiFi buffer (50 mM Tris-HCl, pH 7.5, 70mM NH4Cl, 30 mM KCl, 3.5 mM MgCl2, 0.5 mM spermidine, 8mM putrescine, 2 mM DTT, 3.5 mM MgCl2)
pH: 7.5
Support film200 mesh
VitrificationInstrument: FEI VITROBOT / Cryogen name: NITROGEN / Temperature: 80 K / Humidity: 90 % / Method: blot for 3 seconds / Details: Vitrification instrument: Vitrobot

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F30 / Date: May 1, 2007
Details: automated data collection system AutoEMation (CCD mag. 100000x)
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 25 e/Å2 / Electron beam tilt params: no tilt / Illumination mode: FLOOD BEAM
LensMagnification: 59000 X (nominal), 58269 X (calibrated)
Astigmatism: objective corrected at 100,000 times magnification
Cs: 2.26 mm / Imaging mode: BRIGHT FIELD / Defocus: 1200 - 4000 nm / Energy filter: no filter
Specimen HolderHolder: cartridge / Model: OTHER / Temperature: 80.7 K ( 80.7 - 80.7 K)
CameraDetector: TVIPS TEMCAM-F415 (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 37000
3D reconstructionSoftware: XMIPP, SPIDER / CTF correction: defocus groups, Wiener filter / Details: Classification using ML3D / Resolution: 11.5 Å / Resolution method: FSC 0.5

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Atomic model buiding

Modeling #1Software: MDFF / Refinement protocol: flexible / Target criteria: RMSD, cross correlation / Refinement space: REAL
Details: Protocol: Molecular Dynamics based flexible fitting
Input PDB model: 2I2U
Modeling #2Software: MDFF / Refinement protocol: flexible / Target criteria: RMSD, cross correlation / Refinement space: REAL
Details: Protocol: Molecular Dynamics based flexible fitting
Input PDB model: 2I2V
Output model

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