EMDB-5800: Structure of the Ribosome with Elongation Factor G Trapped in the...

Basic information

• Entry: Database: EMDB / ID: EMD-5800
• Title: Structure of the Ribosome with Elongation Factor G Trapped in the Pre-Translocation State
• Map data: Reconstruction of a pre-translocation ribosome with EF-G bound

Sample

• Sample: Pre-translocation 70S ribosome with bound EF-G and bound A/P* and P/E hybrid state tRNA
• Complex: 70S ribosome
• Protein or peptide: Elongation Factor G
• RNA: Transfer RNA
• RNA: Messenger RNA
• Ligand: Viomycin

• Keywords: protein structure / translation / EF-G / electron cryo-microscopy / single particle analysis

Function / homology

Function:

intracellular anatomical structure / ribosome disassembly / guanosine tetraphosphate binding / stringent response / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / RNA folding / translational elongation / transcriptional attenuation / translation elongation factor activity / positive regulation of ribosome biogenesis / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / translational termination / negative regulation of cytoplasmic translation / four-way junction DNA binding / DnaA-L2 complex / translation repressor activity / negative regulation of translational initiation / regulation of mRNA stability / 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 / transcription elongation factor complex / regulation of DNA-templated transcription elongation / cytosolic ribosome assembly / response to reactive oxygen species / DNA endonuclease activity / transcription antitermination / regulation of cell growth / DNA-templated transcription termination / response to radiation / maintenance of translational fidelity / mRNA 5'-UTR binding / ribosome biogenesis / regulation of translation / large ribosomal subunit / transferase activity / ribosome binding / ribosomal small subunit biogenesis / ribosomal small subunit assembly / small ribosomal subunit / 5S rRNA binding / ribosomal large subunit assembly / cytosolic small ribosomal subunit / large ribosomal subunit rRNA binding / small ribosomal subunit rRNA binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / cytosolic large ribosomal subunit / cytoplasmic translation / tRNA binding / negative regulation of translation / rRNA binding / ribosome / structural constituent of ribosome / translation / response to antibiotic / negative regulation of DNA-templated transcription / GTPase activity / mRNA binding / GTP binding / protein homodimerization activity / DNA binding / RNA binding / zinc ion binding / membrane / cytosol / cytoplasm

Domain/homology:

Translation elongation factor EFG/EF2 / Translation elongation factor EFG/EF2 / : / Elongation factor G, domain III / EFG, domain V / Ribosomal protein L7/L12, oligomerisation / Ribosomal protein L7/L12, oligomerisation domain superfamily / Ribosomal protein L7/L12 dimerisation domain / Ribosomal protein L7/L12 / Ribosomal protein L7/L12, C-terminal / Ribosomal protein L7/L12 C-terminal domain / Ribosomal protein L7/L12, C-terminal/adaptor protein ClpS-like / Ribosomal protein L1, bacterial-type / Elongation Factor G, domain II / Elongation Factor G, domain III / Ribosomal protein L10, eubacterial, conserved site / Ribosomal protein L10 signature. / Translation elongation factor EFG/EF2, domain IV / Elongation factor G, domain IV / Elongation factor G, domain IV / Elongation factor G C-terminus / Ribosomal protein L10 / Elongation factor EFG, domain V-like / Elongation factor G C-terminus / EF-G domain III/V-like / Tr-type G domain, conserved site / Translational (tr)-type guanine nucleotide-binding (G) domain signature. / : / Ribosomal protein L1, conserved site / Ribosomal protein L1 signature. / Ribosomal protein L1 / Translation elongation factor EFTu-like, domain 2 / Ribosomal protein L1, 3-layer alpha/beta-sandwich / Ribosomal protein S21, conserved site / Ribosomal protein S21 signature. / Ribosomal protein L11, bacterial-type / Elongation factor Tu domain 2 / Ribosomal protein L25, short-form / Ribosomal protein L1-like / Ribosomal protein L1/ribosomal biogenesis protein / Ribosomal protein L1p/L10e family / Ribosomal protein S14, bacterial/plastid / Ribosomal protein S21 superfamily / Ribosomal protein S16, conserved site / Ribosomal protein S16 signature. / Ribosomal protein S21 / Translational (tr)-type GTP-binding domain / Elongation factor Tu GTP binding domain / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / 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 L17 signature. / 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 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 L5, bacterial-type / Ribosomal protein L18, bacterial-type / : / Ribosomal protein S19, bacterial-type

Component:

Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL15 / Elongation factor G / Large ribosomal subunit protein uL10 / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein bL12 / 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 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 / Small ribosomal subunit protein uS15 / 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

• Biological species: Escherichia coli (E. coli) / synthetic construct (others) / unidentified (others)
• Method: single particle reconstruction / cryo EM / Resolution: 7.6 Å
• Authors: Brilot AF / Korostelev AA / Ermolenko DN / Grigorieff N
• Citation: Journal: Proc Natl Acad Sci U S A / Year: 2013; Title: Structure of the ribosome with elongation factor G trapped in the pretranslocation state.; Authors: Axel F Brilot / Andrei A Korostelev / Dmitri N Ermolenko / Nikolaus Grigorieff / ; Abstract: During protein synthesis, tRNAs and their associated mRNA codons move sequentially on the ribosome from the A (aminoacyl) site to the P (peptidyl) site to the E (exit) site in a process catalyzed by a universally conserved ribosome-dependent GTPase [elongation factor G (EF-G) in prokaryotes and elongation factor 2 (EF-2) in eukaryotes]. Although the high-resolution structure of EF-G bound to the posttranslocation ribosome has been determined, the pretranslocation conformation of the ribosome bound with EF-G and A-site tRNA has evaded visualization owing to the transient nature of this state. Here we use electron cryomicroscopy to determine the structure of the 70S ribosome with EF-G, which is trapped in the pretranslocation state using antibiotic viomycin. Comparison with the posttranslocation ribosome shows that the small subunit of the pretranslocation ribosome is rotated by ∼12° relative to the large subunit. Domain IV of EF-G is positioned in the cleft between the body and head of the small subunit outwardly of the A site and contacts the A-site tRNA. Our findings suggest a model in which domain IV of EF-G promotes the translocation of tRNA from the A to the P site as the small ribosome subunit spontaneously rotates back from the hybrid, rotated state into the nonrotated posttranslocation state.

History

Deposition	Nov 19, 2013	-
Header (metadata) release	Dec 18, 2013	-
Map release	Dec 18, 2013	-
Update	Jul 23, 2014	-
Current status	Jul 23, 2014	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_5800.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Reconstruction of a pre-translocation ribosome with EF-G bound
• Voxel size: X=Y=Z: 1.04 Å

Density

Contour Level	By AUTHOR: 0.32 / Movie #1: 0.32
Minimum - Maximum	-0.45880854 - 1.44600463
Average (Standard dev.)	-0.02902501 (±0.17701891)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 320 320 320 Spacing 320 320 320
Cell	A=B=C: 332.8 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	1.04	1.04	1.04
M x/y/z	320	320	320
origin x/y/z	0.000	0.000	0.000
length x/y/z	332.800	332.800	332.800
α/β/γ	90.000	90.000	90.000
start NX/NY/NZ	-95	-75	153
NX/NY/NZ	200	200	200
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	320	320	320
D min/max/mean	-0.459	1.446	-0.029

Supplemental data

Sample components

Entire : Pre-translocation 70S ribosome with bound EF-G and bound A/P* and...

• Entire: Name: Pre-translocation 70S ribosome with bound EF-G and bound A/P* and P/E hybrid state tRNA

Components

• Sample: Pre-translocation 70S ribosome with bound EF-G and bound A/P* and P/E hybrid state tRNA
• Complex: 70S ribosome
• Protein or peptide: Elongation Factor G
• RNA: Transfer RNA
• RNA: Messenger RNA
• Ligand: Viomycin

Supramolecule #1000: Pre-translocation 70S ribosome with bound EF-G and bound A/P* and...

• Supramolecule: Name: Pre-translocation 70S ribosome with bound EF-G and bound A/P* and P/E hybrid state tRNA; type: sample / ID: 1000 / Details: Sample was monodisperse / Number unique components: 5
• Molecular weight: Theoretical: 3 MDa

Supramolecule #1: 70S ribosome

• Supramolecule: Name: 70S ribosome / type: complex / ID: 1 / Recombinant expression: No / Database: NCBI / Ribosome-details: ribosome-prokaryote: ALL
• Source (natural): Organism: Escherichia coli (E. coli) / Strain: MRE600
• Molecular weight: Theoretical: 3 MDa

Macromolecule #1: Elongation Factor G

• Macromolecule: Name: Elongation Factor G / type: protein_or_peptide / ID: 1 / Name.synonym: EF-G / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
• Source (natural): Organism: Escherichia coli (E. coli) / Strain: K-12
• Molecular weight: Theoretical: 78 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: pET24b
• Sequence: UniProtKB: Elongation factor G; GO: translational elongation, GTP binding, translation elongation factor activity, intracellular anatomical structure; InterPro: Translation elongation factor EFG/EF2

Macromolecule #2: Transfer RNA

• Macromolecule: Name: Transfer RNA / type: rna / ID: 2 / Name.synonym: tRNA / Details: Two tRNA (P and E site) / Classification: TRANSFER / Structure: DOUBLE HELIX / Synthetic?: No
• Source (natural): Organism: Escherichia coli (E. coli) / Strain: K-12
• Molecular weight: Experimental: 25 KDa

Macromolecule #3: Messenger RNA

• Macromolecule: Name: Messenger RNA / type: rna / ID: 3 / Name.synonym: mRNA / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: Yes
• Source (natural): Organism: synthetic construct (others)
• Molecular weight: Theoretical: 12 KDa

Sequence

String:

GGCAAGGAGG UAAAAAUGUU UAAACGUAAA UCUACU

Macromolecule #4: Viomycin

• Macromolecule: Name: Viomycin / type: ligand / ID: 4 / Name.synonym: Vio / Number of copies: 1 / Recombinant expression: No / Database: NCBI
• Source (natural): Organism: unidentified (others)
• Molecular weight: Theoretical: 1 KDa

Chemical component information

ChemComp, No image

ChemComp-PRD_000226:
Unknown entry

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 0.4 mg/mL
• Buffer: pH: 7.6 ; Details: 10 mM HEPES-KOH, 5 mM MgCl2, 90 mM NH4Cl, 2 mM spermidine, 0.1 mM spermine, 6 mM BME, 0.5 mM viomycin, 0.5 mM GTP, 0.5 mM fusidic acid
• Grid: Details: C-flat 1.2/1.3 holey carbon 400 mesh copper grid, glow discharged with a current of -20 mA for 45 seconds in an EMITECH K100X glow discharge unit
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 95 % / Instrument: FEI VITROBOT MARK II; Method: 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.

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Alignment procedure: Legacy - Astigmatism: Automatically corrected using FEI software
• Date: Nov 2, 2012
• Image recording: Category: CCD / Film or detector model: FEI FALCON I (4k x 4k) / Digitization - Sampling interval: 14.0 µm / Number real images: 13341 / Average electron dose: 30 e/Ų / Bits/pixel: 16
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Calibrated magnification: 134615 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.01 mm / Nominal defocus max: 6.95 µm / Nominal defocus min: 1.15 µm / Nominal magnification: 133333
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Details: Refinement and 3D classification performed by Frealign. See primary citation Supplementary Information for details.
• CTF correction: Details: CTFFIND3, FREALIGN per micrograph
• Final reconstruction: Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 7.6 Å / Resolution method: OTHER / Software - Name: EMAN2, IMAGIC, FREALIGN, RSAMPLE, CTFFIND3; Details: Refinement included data to 12 Angstrom resolution to limit FSC bias. See primary citation Supplementary Information for details.; Number images used: 85115

Atomic model buiding 1

Initial model

PDB ID:

4gd1
PDB Unreleased entry

• Software: Name: Chimera, CNS
• Details: Chain Y not used in refinement. See primary citation Supplementary Information for details.
• Refinement: Space: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation

Output model

PDB-4v7d:
Structure of the Ribosome with Elongation Factor G Trapped in the Pre-Translocation State (pre-translocation 70S*tRNA*EF-G structure)

Atomic model buiding 2

Initial model

PDB ID:

3r8s
PDB Unreleased entry

• Software: Name: Chimera, CNS
• Details: See primary citation Supplementary Information for details.
• Refinement: Space: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation

Output model

PDB-4v7d:
Structure of the Ribosome with Elongation Factor G Trapped in the Pre-Translocation State (pre-translocation 70S*tRNA*EF-G structure)

Atomic model buiding 3

Initial model

PDB ID:

2bm0

• Software: Name: Chimera, CNS
• Details: A homology model of E. coli EF-G was created using the SWISS-MODEL from T. thermophilus EF-G. See primary citation Supplementary Information for details.
• Refinement: Space: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation

Output model

PDB-4v7d:
Structure of the Ribosome with Elongation Factor G Trapped in the Pre-Translocation State (pre-translocation 70S*tRNA*EF-G structure)

Atomic model buiding 4

Initial model

PDB ID:

1mms

• Software: Name: Chimera, CNS
• Details: Used to rebuild L11 stalk.
• Refinement: Space: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation

Output model

PDB-4v7d:
Structure of the Ribosome with Elongation Factor G Trapped in the Pre-Translocation State (pre-translocation 70S*tRNA*EF-G structure)

Atomic model buiding 5

Initial model

PDB ID:

1dd3

• Software: Name: Chimera, CNS
• Details: Used to rebuild L12.
• Refinement: Space: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation

Output model

PDB-4v7d:
Structure of the Ribosome with Elongation Factor G Trapped in the Pre-Translocation State (pre-translocation 70S*tRNA*EF-G structure)

Atomic model buiding 6

Initial model

PDB ID:

3u4m

• Software: Name: Chimera, CNS
• Details: Used to build homology model for L1.
• Refinement: Space: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation

Output model

PDB-4v7d:
Structure of the Ribosome with Elongation Factor G Trapped in the Pre-Translocation State (pre-translocation 70S*tRNA*EF-G structure)

Atomic model buiding 7

Initial model

PDB ID:

1zav

• Software: Name: Chimera, CNS
• Details: Used to build homology model for L10.
• Refinement: Space: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation

Output model

PDB-4v7d:
Structure of the Ribosome with Elongation Factor G Trapped in the Pre-Translocation State (pre-translocation 70S*tRNA*EF-G structure)