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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: EMD-5362
TitleNew mRNA-tRNA translocation intermediates revealed by cryoEM: Class 6 (rotated 70S ribosome with A-site and P-site tRNAs)
Map dataCryo-EM-revealed rotated 70S ribosome with A and P site tRNAs (class 6)
Sample
  • Sample: Trp-tRNA-EFTu-GDP-kir-70S ribosome
  • Complex: 70S ribosome
Keywordsribosome / 70S / rotated / intermediates / translocation / tRNA
Function / homology
Function and homology information


negative regulation of cytoplasmic translational initiation / stringent response / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity ...negative regulation of cytoplasmic translational initiation / stringent response / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis / translational termination / negative regulation of cytoplasmic translation / four-way junction DNA binding / DnaA-L2 complex / translation repressor activity / regulation of mRNA stability / negative regulation of translational initiation / negative regulation of DNA-templated DNA replication initiation / mRNA regulatory element binding translation repressor activity / assembly of large subunit precursor of preribosome / positive regulation of RNA splicing / ribosome assembly / regulation of DNA-templated transcription elongation / transcription elongation factor complex / cytosolic ribosome assembly / response to reactive oxygen species / DNA endonuclease activity / transcription antitermination / translational initiation / regulation of cell growth / DNA-templated transcription termination / response to radiation / maintenance of translational fidelity / mRNA 5'-UTR binding / ribosome biogenesis / large ribosomal subunit / regulation of translation / ribosome binding / transferase activity / ribosomal small subunit biogenesis / ribosomal small subunit assembly / small ribosomal subunit / small ribosomal subunit rRNA binding / 5S rRNA binding / ribosomal large subunit assembly / cytosolic small ribosomal subunit / large ribosomal subunit rRNA binding / cytosolic large ribosomal subunit / cytoplasmic translation / tRNA binding / negative regulation of translation / rRNA binding / structural constituent of ribosome / ribosome / translation / response to antibiotic / negative regulation of DNA-templated transcription / mRNA binding / DNA binding / RNA binding / zinc ion binding / membrane / cytosol / cytoplasm
Similarity search - Function
Ribosomal protein L1, bacterial-type / Ribosomal protein L10, eubacterial, conserved site / Ribosomal protein L10 signature. / Ribosomal protein L10 / : / Ribosomal protein L1, conserved site / Ribosomal protein L1 signature. / Ribosomal protein L1 / Ribosomal protein L1, 3-layer alpha/beta-sandwich / Ribosomal protein L1-like ...Ribosomal protein L1, bacterial-type / Ribosomal protein L10, eubacterial, conserved site / Ribosomal protein L10 signature. / Ribosomal protein L10 / : / Ribosomal protein L1, conserved site / Ribosomal protein L1 signature. / Ribosomal protein L1 / Ribosomal protein L1, 3-layer alpha/beta-sandwich / Ribosomal protein L1-like / Ribosomal protein L1/ribosomal biogenesis protein / Ribosomal protein L1p/L10e family / Ribosomal protein L11, bacterial-type / Ribosomal protein S21, conserved site / Ribosomal protein S21 signature. / Ribosomal protein L25, short-form / Ribosomal protein S14, bacterial/plastid / Ribosomal protein L31 type A / Ribosomal protein S21 superfamily / Ribosomal protein S21 / Ribosomal protein S16, conserved site / Ribosomal protein S16 signature. / Ribosomal protein L31 signature. / Ribosomal protein L31 / Ribosomal protein L31 superfamily / Ribosomal protein L31 / Ribosomal protein S21 / Ribosomal protein L11, conserved site / Ribosomal protein L11 signature. / Ribosomal protein L10-like domain superfamily / Ribosomal protein L10P / Ribosomal protein L10 / Ribosomal protein L16 signature 1. / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / Ribosomal protein L16 signature 2. / Ribosomal protein L16, conserved site / Ribosomal protein L6, conserved site / Ribosomal protein L6 signature 1. / : / Ribosomal protein L9 signature. / Ribosomal protein L9, bacteria/chloroplast / Ribosomal protein L9, C-terminal / Ribosomal protein L9, C-terminal domain / Ribosomal protein L9, C-terminal domain superfamily / Ribosomal protein L11, N-terminal / Ribosomal protein L11, N-terminal domain / Ribosomal protein L11/L12 / Ribosomal protein L11, C-terminal / Ribosomal protein L11, C-terminal domain superfamily / Ribosomal protein L11/L12, N-terminal domain superfamily / Ribosomal protein L11/L12 / Ribosomal protein L11, RNA binding domain / Ribosomal protein L17 signature. / Ribosomal L25p family / Ribosomal protein L25 / Ribosomal protein L36 signature. / Ribosomal protein L32p, bacterial type / Ribosomal protein L28/L24 superfamily / Ribosomal protein L25/Gln-tRNA synthetase, N-terminal / Ribosomal protein L25/Gln-tRNA synthetase, anti-codon-binding domain superfamily / : / Ribosomal protein L9, N-terminal domain superfamily / Ribosomal protein L9 / Ribosomal protein L9, N-terminal / Ribosomal protein L9, N-terminal domain / Ribosomal protein L33, conserved site / Ribosomal protein L33 signature. / Ribosomal protein L35, conserved site / Ribosomal protein L35 signature. / Ribosomal protein L28 / Ribosomal protein L35, non-mitochondrial / Ribosomal protein L18, bacterial-type / : / Ribosomal protein L6, bacterial-type / Ribosomal protein S3, bacterial-type / Ribosomal protein S19, bacterial-type / Ribosomal protein S13, bacterial-type / Ribosomal protein S6, conserved site / Ribosomal protein S6 signature. / Ribosomal protein L9/RNase H1, N-terminal / Ribosomal protein S7, bacterial/organellar-type / Ribosomal protein S11, bacterial-type / Ribosomal protein S20 / Ribosomal protein S20 superfamily / Ribosomal protein S20 / Ribosomal protein L5, bacterial-type / Ribosomal protein L36 / Ribosomal protein L36 superfamily / Ribosomal protein L36 / Ribosomal protein S4, bacterial-type / Ribosomal protein L19, conserved site / Ribosomal protein L19 signature. / 30S ribosomal protein S17 / Ribosomal protein S5, bacterial-type / Ribosomal protein L27, conserved site / Ribosomal protein L27 signature. / Ribosomal protein S6, plastid/chloroplast / Ribosomal protein L20 signature. / Ribosomal protein L22, bacterial/chloroplast-type
Similarity search - Domain/homology
Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein uL10 / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein uL1 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL28 ...Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein uL10 / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein uL1 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL28 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein bL31 / Large ribosomal subunit protein bL32 / Large ribosomal subunit protein bL33 / Large ribosomal subunit protein bL34 / Large ribosomal subunit protein bL35 / Large ribosomal subunit protein bL36A / Large ribosomal subunit protein bL9 / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein uS11 / Small ribosomal subunit protein uS12 / Small ribosomal subunit protein uS13 / Small ribosomal subunit protein bS16 / Small ribosomal subunit protein bS18 / Small ribosomal subunit protein uS19 / Small ribosomal subunit protein bS20 / Small ribosomal subunit protein uS2 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS4 / Small ribosomal subunit protein uS5 / Small ribosomal subunit protein uS8 / Small ribosomal subunit protein uS9 / Large ribosomal subunit protein uL13 / Large ribosomal subunit protein uL14 / Large ribosomal subunit protein uL16 / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein bL17 / Large ribosomal subunit protein bL21 / Large ribosomal subunit protein uL30 / Large ribosomal subunit protein uL6 / Small ribosomal subunit protein uS14 / Small ribosomal subunit protein uS17 / Large ribosomal subunit protein uL18 / Large ribosomal subunit protein uL2 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein uL24 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL22 / Large ribosomal subunit protein uL5 / Small ribosomal subunit protein bS21 / Large ribosomal subunit protein bL25 / Small ribosomal subunit protein uS15
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 11.5 Å
AuthorsAgirrezabala X / Liao HY / Schreiner E / Fu J / Ortiz-Meoz RF / Schulten K / Green R / Frank J
CitationJournal: Proc Natl Acad Sci U S A / Year: 2012
Title: Structural characterization of mRNA-tRNA translocation intermediates.
Authors: Xabier Agirrezabala / Hstau Y Liao / Eduard Schreiner / Jie Fu / Rodrigo F Ortiz-Meoz / Klaus Schulten / Rachel Green / Joachim Frank /
Abstract: Cryo-EM analysis of a wild-type Escherichia coli pretranslocational sample has revealed the presence of previously unseen intermediate substates of the bacterial ribosome during the first phase of ...Cryo-EM analysis of a wild-type Escherichia coli pretranslocational sample has revealed the presence of previously unseen intermediate substates of the bacterial ribosome during the first phase of translocation, characterized by intermediate intersubunit rotations, L1 stalk positions, and tRNA configurations. Furthermore, we describe the domain rearrangements in quantitative terms, which has allowed us to characterize the processivity and coordination of the conformational reorganization of the ribosome, along with the associated changes in tRNA ribosome-binding configuration. The results are consistent with the view of the ribosome as a molecular machine employing Brownian motion to reach a functionally productive state via a series of substates with incremental changes in conformation.
History
DepositionDec 2, 2011-
Header (metadata) releaseJan 26, 2012-
Map releaseFeb 16, 2012-
UpdateJul 23, 2014-
Current statusJul 23, 2014Processing site: RCSB / Status: Released

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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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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5362_1.gif

Downloads & links

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Map

FileDownload / File: emd_5362.map.gz / Format: CCP4 / Size: 58.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM-revealed rotated 70S ribosome with A and P site tRNAs (class 6)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.5 Å/pix.
x 250 pix.
= 375. Å		1.5 Å/pix.
x 250 pix.
= 375. Å		1.5 Å/pix.
x 250 pix.
= 375. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.5 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1 / Movie #1: 0.15
Minimum - Maximum-0.49196333 - 1.57512212
Average (Standard dev.)0.02891555 (±0.20386297)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-124-124-124
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-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
Components
  • Sample: Trp-tRNA-EFTu-GDP-kir-70S ribosome
  • Complex: 70S ribosome

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Supramolecule #1000: Trp-tRNA-EFTu-GDP-kir-70S ribosome

SupramoleculeName: Trp-tRNA-EFTu-GDP-kir-70S ribosome / type: sample / ID: 1000 / Oligomeric state: monomeric / Number unique components: 3
Molecular weightExperimental: 2.8 MDa / Theoretical: 2.8 MDa

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Supramolecule #1: 70S ribosome

SupramoleculeName: 70S ribosome / type: complex / ID: 1 / Recombinant expression: No / Database: NCBI / Ribosome-details: ribosome-prokaryote: ALL
Source (natural)Organism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.03 mg/mL
BufferpH: 7.5
Details: 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)
GridDetails: 200 mesh
VitrificationCryogen name: NITROGEN / Chamber humidity: 90 % / Chamber temperature: 80 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot / Method: blot for 3 seconds

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Electron microscopy

MicroscopeFEI TECNAI F30
TemperatureMin: 80.7 K / Max: 80.7 K / Average: 80.7 K
Alignment procedureLegacy - Astigmatism: objective corrected at 100,000 times magnification
Legacy - Electron beam tilt params: no tilt
Specialist opticsEnergy filter - Name: no filter
Detailsautomated data collection system AutoEMation (CCD mag. 100000x)
DateMay 1, 2007
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Average electron dose: 25 e/Å2
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 58269 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.26 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 59000
Sample stageSpecimen holder: cartridge / Specimen holder model: OTHER
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: defocus groups, Wiener filter
Final reconstructionResolution.type: BY AUTHOR / Resolution: 11.5 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: XMIPP, SPIDER / Details: Classification using ML3D / Number images used: 37000

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Atomic model buiding 1

Initial modelPDB ID:

2i2u
PDB Unreleased entry

SoftwareName: MDFF
DetailsProtocol: Molecular Dynamics based flexible fitting
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: RMSD, cross correlation
Output model

PDB-4v6r:
Structural characterization of mRNA-tRNA translocation intermediates (class 6 of the six classes)

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Atomic model buiding 2

Initial modelPDB ID:

2i2v
PDB Unreleased entry

SoftwareName: MDFF
DetailsProtocol: Molecular Dynamics based flexible fitting
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: RMSD, cross correlation
Output model

PDB-4v6r:
Structural characterization of mRNA-tRNA translocation intermediates (class 6 of the six classes)

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