+Open data
-Basic information
Entry | Database: PDB / ID: 5u9f | |||||||||
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Title | 3.2 A cryo-EM ArfA-RF2 ribosome rescue complex (Structure II) | |||||||||
Components |
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Keywords | RIBOSOME / ARFA RF2 ribosome rescue complex | |||||||||
Function / homology | Function and homology information translation release factor activity, codon specific / negative regulation of cytoplasmic translational initiation / stringent response / ribosomal large subunit binding / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation ...translation release factor activity, codon specific / negative regulation of cytoplasmic translational initiation / stringent response / ribosomal large subunit binding / 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 / negative regulation of cytoplasmic translation / four-way junction DNA binding / translational termination / DnaA-L2 complex / translation repressor activity / negative regulation of translational initiation / negative regulation of DNA-templated DNA replication initiation / regulation of mRNA stability / rescue of stalled ribosome / mRNA regulatory element binding translation repressor activity / ribosome assembly / positive regulation of RNA splicing / assembly of large subunit precursor of preribosome / transcription elongation factor complex / cytosolic ribosome assembly / regulation of DNA-templated transcription elongation / DNA endonuclease activity / ribosomal large subunit assembly / transcription antitermination / response to reactive oxygen species / translational initiation / regulation of cell growth / DNA-templated transcription termination / maintenance of translational fidelity / response to radiation / mRNA 5'-UTR binding / large ribosomal subunit / ribosome biogenesis / ribosome binding / regulation of translation / ribosomal small subunit biogenesis / ribosomal small subunit assembly / small ribosomal subunit / small ribosomal subunit rRNA binding / transferase activity / 5S rRNA binding / large ribosomal subunit rRNA binding / cytosolic small ribosomal subunit / cytosolic large ribosomal subunit / cytoplasmic translation / tRNA binding / molecular adaptor activity / rRNA binding / negative regulation of translation / ribosome / structural constituent of ribosome / translation / ribonucleoprotein complex / response to antibiotic / negative regulation of DNA-templated transcription / mRNA binding / DNA binding / RNA binding / zinc ion binding / membrane / metal ion binding / cytoplasm / cytosol Similarity search - Function | |||||||||
Biological species | Escherichia coli (E. coli) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å | |||||||||
Authors | Demo, G. / Svidritskiy, E. / Madireddy, R. / Diaz-Avalos, R. / Grant, T. / Grigorieff, N. / Sousa, D. / Korostelev, A.A. | |||||||||
Funding support | United States, 2items
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Citation | Journal: Elife / Year: 2017 Title: Mechanism of ribosome rescue by ArfA and RF2. Authors: Gabriel Demo / Egor Svidritskiy / Rohini Madireddy / Ruben Diaz-Avalos / Timothy Grant / Nikolaus Grigorieff / Duncan Sousa / Andrei A Korostelev / Abstract: ArfA rescues ribosomes stalled on truncated mRNAs by recruiting release factor RF2, which normally binds stop codons to catalyze peptide release. We report two 3.2 Å resolution cryo-EM structures - ...ArfA rescues ribosomes stalled on truncated mRNAs by recruiting release factor RF2, which normally binds stop codons to catalyze peptide release. We report two 3.2 Å resolution cryo-EM structures - determined from a single sample - of the 70S ribosome with ArfA•RF2 in the A site. In both states, the ArfA C-terminus occupies the mRNA tunnel downstream of the A site. One state contains a compact inactive RF2 conformation. Ordering of the ArfA N-terminus in the second state rearranges RF2 into an extended conformation that docks the catalytic GGQ motif into the peptidyl-transferase center. Our work thus reveals the structural dynamics of ribosome rescue. The structures demonstrate how ArfA 'senses' the vacant mRNA tunnel and activates RF2 to mediate peptide release without a stop codon, allowing stalled ribosomes to be recycled. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 5u9f.cif.gz | 3.8 MB | Display | PDBx/mmCIF format |
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PDB format | pdb5u9f.ent.gz | Display | PDB format | |
PDBx/mmJSON format | 5u9f.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/u9/5u9f ftp://data.pdbj.org/pub/pdb/validation_reports/u9/5u9f | HTTPS FTP |
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-Related structure data
Related structure data | 8521MC 8522C 5u9gC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-RNA chain , 5 types, 6 molecules A0102XWV
#1: RNA chain | Mass: 498725.406 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: GenBank: 944404559 | ||
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#2: RNA chain | Mass: 941322.188 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: GenBank: 42756 | ||
#3: RNA chain | Mass: 38483.926 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: GenBank: 1072784372 | ||
#5: RNA chain | Mass: 24802.785 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: GenBank: 1114191980 #38: RNA chain | | Mass: 4597.844 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 |
-Protein , 2 types, 2 molecules YZ
#4: Protein | Mass: 8190.438 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12 / Gene: arfA, yhdL, b4550, JW3253 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P36675 |
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#39: Protein | Mass: 41300.660 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12 / Gene: prfB, supK, b2891, JW5847 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P07012 |
+50S ribosomal protein ... , 32 types, 32 molecules 0304050607080910111213141516171819202122232425262728293031323334
-30S ribosomal protein ... , 20 types, 20 molecules BCDEFGHIJKLMNOPQRSTU
#40: Protein | Mass: 26781.670 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: D7ZK99, UniProt: P0A7V0*PLUS |
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#41: Protein | Mass: 26031.316 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: B7MCS9, UniProt: P0A7V3*PLUS |
#42: Protein | Mass: 23514.199 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: D7Z9H7, UniProt: P0A7V8*PLUS |
#43: Protein | Mass: 17629.398 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: D7Z9H2, UniProt: P0A7W1*PLUS |
#44: Protein | Mass: 15211.058 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: D7ZI17, UniProt: P02358*PLUS |
#45: Protein | Mass: 17637.445 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: D7ZAN1, UniProt: P02359*PLUS |
#46: Protein | Mass: 14146.557 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: D7Z9G9, UniProt: P0A7W7*PLUS |
#47: Protein | Mass: 14886.270 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: D7ZES9, UniProt: P0A7X3*PLUS |
#48: Protein | Mass: 11755.597 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: D7Z9F4, UniProt: P0A7R5*PLUS |
#49: Protein | Mass: 13870.975 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: B7MCR3, UniProt: P0A7R9*PLUS |
#50: Protein | Mass: 13768.157 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: D7ZAN0, UniProt: P0A7S3*PLUS |
#51: Protein | Mass: 13128.467 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: H4UQ02, UniProt: P0A7S9*PLUS |
#52: Protein | Mass: 11606.560 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: D7Z9G8, UniProt: P0AG59*PLUS |
#53: Protein | Mass: 10290.816 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: D7ZEL4, UniProt: P0ADZ4*PLUS |
#54: Protein | Mass: 9207.572 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: B7MIU7, UniProt: P0A7T3*PLUS |
#55: Protein | Mass: 9724.491 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: D7Z9G4, UniProt: P0AG63*PLUS |
#56: Protein | Mass: 9005.472 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: A0A0E2KXL3, UniProt: P0A7T7*PLUS |
#57: Protein | Mass: 10455.355 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: D7Z9F9, UniProt: P0A7U3*PLUS |
#58: Protein | Mass: 9708.464 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: D7ZAS2, UniProt: P0A7U7*PLUS |
#59: Protein | Mass: 8524.039 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE600 / References: UniProt: A0A0E2L2J1, UniProt: P68679*PLUS |
-Non-polymers , 2 types, 361 molecules
#60: Chemical | ChemComp-MG / #61: Chemical | ChemComp-ZN / | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: ArfA-RF2 ribosome rescue complex / Type: RIBOSOME / Entity ID: #1-#59 / Source: MULTIPLE SOURCES | ||||||||||||||||||||||||||||||
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Molecular weight | Value: 2.5 MDa / Experimental value: NO | ||||||||||||||||||||||||||||||
Source (natural) | Organism: Escherichia coli (E. coli) / Strain: MRE600 | ||||||||||||||||||||||||||||||
Source (recombinant) | Organism: Escherichia coli (E. coli) / Strain: K-12 | ||||||||||||||||||||||||||||||
Buffer solution | pH: 7 | ||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||||
Specimen support | Details: Using a Solarus 950 plasma cleaning system. The forward RF target was set to 7w. Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: C-flat-2/2 | ||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 29000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 1.2 e/Å2 / Detector mode: COUNTING / Film or detector model: DIRECT ELECTRON DE-20 (5k x 3k) / Num. of grids imaged: 1 / Num. of real images: 3760 |
Image scans | Movie frames/image: 54 / Used frames/image: 2-27 |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 539311 | ||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 96070 / Num. of class averages: 1 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 100 / Protocol: OTHER / Space: REAL / Target criteria: Correlation coefficient |