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- EMDB-1541: Visualization of the hybrid state of tRNA binding promoted by spo... -

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Basic information

Entry
Database: EMDB / ID: EMD-1541
TitleVisualization of the hybrid state of tRNA binding promoted by spontaneous ratcheting of the ribosome
Map dataHybrid configuration pre-translocational ribosome
Sample
  • Sample: Hybrid state pre-translocational ribosome
  • Complex: 70S prokaryote ribosome
KeywordsHybrid state tRNA configuration / pre-translocational ribosome / spontaneous ratchet motion
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 8.9 Å
AuthorsAgirrezabala X / Lei J / Brunelle JL / Ortiz-Meoz RF / Green R / Frank J
CitationJournal: Mol Cell / Year: 2008
Title: Visualization of the hybrid state of tRNA binding promoted by spontaneous ratcheting of the ribosome.
Authors: Xabier Agirrezabala / Jianlin Lei / Julie L Brunelle / Rodrigo F Ortiz-Meoz / Rachel Green / Joachim Frank /
Abstract: A crucial step in translation is the translocation of tRNAs through the ribosome. In the transition from one canonical site to the other, the tRNAs acquire intermediate configurations, so-called ...A crucial step in translation is the translocation of tRNAs through the ribosome. In the transition from one canonical site to the other, the tRNAs acquire intermediate configurations, so-called hybrid states. At this stage, the small subunit is rotated with respect to the large subunit, and the anticodon stem loops reside in the A and P sites of the small subunit, while the acceptor ends interact with the P and E sites of the large subunit. In this work, by means of cryo-EM and particle classification procedures, we visualize the hybrid state of both A/P and P/E tRNAs in an authentic factor-free ribosome complex during translocation. In addition, we show how the repositioning of the tRNAs goes hand in hand with the change in the interplay between S13, L1 stalk, L5, H68, H69, and H38 that is caused by the ratcheting of the small subunit.
History
DepositionAug 12, 2008-
Header (metadata) releaseAug 18, 2008-
Map releaseAug 27, 2009-
UpdateMay 8, 2013-
Current statusMay 8, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 31
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 31
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1541.gif

Downloads & links

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Map

FileDownload / File: emd_1541.map.gz / Format: CCP4 / Size: 58.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHybrid configuration pre-translocational ribosome
Voxel sizeX=Y=Z: 1.5 Å
Density
Contour LevelBy EMDB: 30.0 / Movie #1: 31
Minimum - Maximum-117.195213319999993 - 293.753967289999991
Average (Standard dev.)5.17587709 (±27.60373306)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 375.0 Å
α=β=γ: 90.0 °

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-120-120-120
NX/NY/NZ240240240
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS250250250
D min/max/mean-117.195293.7545.176

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Supplemental data

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Sample components

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Entire : Hybrid state pre-translocational ribosome

EntireName: Hybrid state pre-translocational ribosome
Components
  • Sample: Hybrid state pre-translocational ribosome
  • Complex: 70S prokaryote ribosome

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Supramolecule #1000: Hybrid state pre-translocational ribosome

SupramoleculeName: Hybrid state pre-translocational ribosome / type: sample / ID: 1000 / Details: HiFi buffer, 3.5 mM Mg2 / Oligomeric state: Monomeric / Number unique components: 3
Molecular weightExperimental: 2.6 MDa / Theoretical: 2.6 MDa / Method: Sedimentation

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Supramolecule #1: 70S prokaryote ribosome

SupramoleculeName: 70S prokaryote ribosome / type: complex / ID: 1 / Name.synonym: Ribosome / Details: Pre-translocational ribosome / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: ALL
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MRE 600
Molecular weightExperimental: 2.6 MDa / Theoretical: 2.6 MDa

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.07 mg/mL
BufferpH: 7.5
Details: HiFi (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)
StainingType: NEGATIVE / Details: Cryo-sample
GridDetails: Quantifoil 2/4
VitrificationCryogen name: NITROGEN / Chamber humidity: 90 % / Chamber temperature: 80 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot / Method: Blot for 6 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F30
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 58269 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.26 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 59000
Specialist opticsEnergy filter - Name: No energy filter
Sample stageSpecimen holder: Cartridge / Specimen holder model: OTHER
TemperatureMin: 80.7 K / Max: 80.7 K / Average: 80.7 K
Alignment procedureLegacy - Astigmatism: Objective corrected at 100,000 times magnification
DetailsAutomated data collection system AutoEMation (CCD mag. 100000x)
DateApr 15, 2007
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Average electron dose: 25 e/Å2
Details: Automated data collection system AutoEMation (CCD mag. 100000x) TVIPS TemCam-F415
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Defocus groups, Wiener filter
Final two d classificationNumber classes: 1
Final angle assignmentDetails: Spider
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.9 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Details: Supervised classification / Number images used: 158969
DetailsSupervised classification

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Atomic model buiding 1

Initial model(PDB ID:

2avy
PDB Unreleased entry

,

2aw4
PDB Unreleased entry

,

2j00
PDB Unreleased entry

,

1gix
PDB Unreleased entry

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1lbk

)
SoftwareName: CNS
DetailsProtocol: Real-space refinement. 1lBK(1989-1996 fragment)
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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