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- EMDB-1045: Cryo-EM reveals an active role for aminoacyl-tRNA in the accommod... -

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Basic information

Entry
Database: EMDB / ID: EMD-1045
TitleCryo-EM reveals an active role for aminoacyl-tRNA in the accommodation process.
Map data70S aa-tRNA-EF-Tu.GDP.kirromycin complex
Sample
  • Sample: 70S aa-tRNA-EF-Tu.GDP.kirromycin complex from E.coli
  • Complex: E.coli 70S ribosome
  • RNA: aa-tRNA
  • Ligand: EF-Tu
  • Ligand: GDP
  • Ligand: kirromycin
  • Ligand: MF-mRNA
Function / homology
Function and homology information


guanyl-nucleotide exchange factor complex / guanosine tetraphosphate binding / translational elongation / translation elongation factor activity / response to antibiotic / GTPase activity / GTP binding / RNA binding / plasma membrane / cytoplasm
Similarity search - Function
Translation elongation factor EFTu/EF1A, bacterial/organelle / Elongation factor Tu, domain 2 / Elongation factor Tu (EF-Tu), GTP-binding domain / Translation elongation factor EFTu/EF1A, C-terminal / Elongation factor Tu C-terminal domain / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal / Tr-type G domain, conserved site / Translational (tr)-type guanine nucleotide-binding (G) domain signature. / Translation elongation factor EFTu-like, domain 2 / Elongation factor Tu domain 2 ...Translation elongation factor EFTu/EF1A, bacterial/organelle / Elongation factor Tu, domain 2 / Elongation factor Tu (EF-Tu), GTP-binding domain / Translation elongation factor EFTu/EF1A, C-terminal / Elongation factor Tu C-terminal domain / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal / Tr-type G domain, conserved site / Translational (tr)-type guanine nucleotide-binding (G) domain signature. / Translation elongation factor EFTu-like, domain 2 / Elongation factor Tu domain 2 / Translational (tr)-type GTP-binding domain / Elongation factor Tu GTP binding domain / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / Small GTP-binding protein domain / Translation protein, beta-barrel domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Elongation factor Tu 1 / Elongation factor Tu 2
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 16.8 Å
AuthorsValle M / Sengupta J / Swami NK / Grassucci RA / Burkhardt N / Nierhaus KH / Agrawal RK / Frank J
CitationJournal: EMBO J / Year: 2002
Title: Cryo-EM reveals an active role for aminoacyl-tRNA in the accommodation process.
Authors: Mikel Valle / Jayati Sengupta / Neil K Swami / Robert A Grassucci / Nils Burkhardt / Knud H Nierhaus / Rajendra K Agrawal / Joachim Frank /
Abstract: During the elongation cycle of protein biosynthesis, the specific amino acid coded for by the mRNA is delivered by a complex that is comprised of the cognate aminoacyl-tRNA, elongation factor Tu and ...During the elongation cycle of protein biosynthesis, the specific amino acid coded for by the mRNA is delivered by a complex that is comprised of the cognate aminoacyl-tRNA, elongation factor Tu and GTP. As this ternary complex binds to the ribosome, the anticodon end of the tRNA reaches the decoding center in the 30S subunit. Here we present the cryo- electron microscopy (EM) study of an Escherichia coli 70S ribosome-bound ternary complex stalled with an antibiotic, kirromycin. In the cryo-EM map the anticodon arm of the tRNA presents a new conformation that appears to facilitate the initial codon-anticodon interaction. Furthermore, the elbow region of the tRNA is seen to contact the GTPase-associated center on the 50S subunit of the ribosome, suggesting an active role of the tRNA in the transmission of the signal prompting the GTP hydrolysis upon codon recognition.
History
DepositionApr 15, 2003-
Header (metadata) releaseMay 12, 2003-
Map releaseJan 6, 2004-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 20
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 20
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-1ls2, PDB-1lu3
  • Surface level: 20
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-1ls2
  • Imaged by Jmol
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-1lu3
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_1045.map.gz / Format: CCP4 / Size: 9.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation70S aa-tRNA-EF-Tu.GDP.kirromycin complex
Voxel sizeX=Y=Z: 2.69 Å
Density
Contour Level1: 48.299999999999997 / Movie #1: 20
Minimum - Maximum-82.174800000000005 - 184.76400000000001
Average (Standard dev.)4.36412 (±20.063500000000001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-68-68-68
Dimensions136136136
Spacing136136136
CellA=B=C: 365.84 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.692.692.69
M x/y/z136136136
origin x/y/z0.0000.0000.000
length x/y/z365.840365.840365.840
α/β/γ90.00090.00090.000
start NX/NY/NZ0052
NX/NY/NZ12812855
MAP C/R/S123
start NC/NR/NS-68-68-68
NC/NR/NS136136136
D min/max/mean-82.175184.7644.364

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Supplemental data

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Sample components

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Entire : 70S aa-tRNA-EF-Tu.GDP.kirromycin complex from E.coli

EntireName: 70S aa-tRNA-EF-Tu.GDP.kirromycin complex from E.coli
Components
  • Sample: 70S aa-tRNA-EF-Tu.GDP.kirromycin complex from E.coli
  • Complex: E.coli 70S ribosome
  • RNA: aa-tRNA
  • Ligand: EF-Tu
  • Ligand: GDP
  • Ligand: kirromycin
  • Ligand: MF-mRNA

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Supramolecule #1000: 70S aa-tRNA-EF-Tu.GDP.kirromycin complex from E.coli

SupramoleculeName: 70S aa-tRNA-EF-Tu.GDP.kirromycin complex from E.coli / type: sample / ID: 1000 / Number unique components: 6
Molecular weightExperimental: 2.5 MDa / Theoretical: 2.5 MDa

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Supramolecule #1: E.coli 70S ribosome

SupramoleculeName: E.coli 70S ribosome / type: complex / ID: 1 / Details: 10 pmol / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: ALL
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: aa-tRNA

MacromoleculeName: aa-tRNA / type: rna / ID: 1
Details: 14 pmol; fMet-tRNA(fMet) at the P site and Phe-tRNA(phe) at the A site
Classification: TRANSFER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #2: EF-Tu

MacromoleculeName: EF-Tu / type: ligand / ID: 2 / Details: 20 pmol / Recombinant expression: No
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #3: GDP

MacromoleculeName: GDP / type: ligand / ID: 3
Details: 8 ul of 2.5mM; [Alpha-(32)P]GTP The radioactivity contributed by the Alpha-(32)P moiety of the GDP served to calculate a ~50% occupancy for the elongation factor.
Recombinant expression: No
Source (natural)Organism: synthetic construct (others)

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Macromolecule #4: kirromycin

MacromoleculeName: kirromycin / type: ligand / ID: 4 / Details: 3 ul of 100 mM; antibiotic / Recombinant expression: No
Source (natural)Organism: synthetic construct (others)
Chemical component information

ChemComp-KIR:
KIRROMYCIN

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Macromolecule #5: MF-mRNA

MacromoleculeName: MF-mRNA / type: ligand / ID: 5 / Details: 15 pmol; M(Met).F(Phe)-mRNA / Recombinant expression: No
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

BufferpH: 7.5
Details: 20 mM HEPES-KOH, 6mM MgCl2, 150mM NH4Cl, 4mM 2-metcaptoethanol, 0.05 mM spermine and 2 mM spermidine
VitrificationCryogen name: ETHANE / Chamber humidity: 30 % / Chamber temperature: 93 K / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: two side blotting plunger
Method: Blot for 2 seconds before plunging

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Electron microscopy

MicroscopeFEI/PHILIPS EM420
Electron beamAcceleration voltage: 100 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 52000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 3.844 µm / Nominal defocus min: 1.041 µm / Nominal magnification: 49000
Sample stageSpecimen holder: Cryo transfer / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 93 K
DetailsMicroscope: Philips EM420 Imaging date: 1997
DateJun 1, 1998
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 14 µm / Number real images: 45 / Average electron dose: 10 e/Å2 / Od range: 1.2 / Bits/pixel: 12

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Image processing

CTF correctionDetails: defocus groups
Final two d classificationNumber classes: 2
Final angle assignmentDetails: SPIDER definition
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 16.8 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER
Details: Final map was calculated from 8 CTF corrected defocus groups
Number images used: 7985

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Atomic model buiding 1

Initial modelPDB ID:
SoftwareName: manual
DetailsProtocol: Rigid Body. The EF-Tu domains were separately fitted by manual docking using program O (pdb codes for fitted coordinates: 1LS2, 1LU3)
RefinementProtocol: RIGID BODY FIT / Target criteria: cross correlation coefficient
Output model

PDB-1ls2:
Fitting of EF-Tu and tRNA in the Low Resolution Cryo-EM Map of an EF-Tu Ternary Complex (GDP and Kirromycin) Bound to E. coli 70S Ribosome

PDB-1lu3:
Separate Fitting of the Anticodon Loop Region of tRNA (nucleotide 26-42) in the Low Resolution Cryo-EM Map of an EF-Tu Ternary Complex (GDP and Kirromycin) Bound to E. coli 70S Ribosome

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