+Open data
-Basic information
Entry | Database: PDB / ID: 5wdt | |||||||||
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Title | 70S ribosome-EF-Tu H84A complex with GppNHp | |||||||||
Components |
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Keywords | RIBOSOME / translation / GTPase | |||||||||
Function / homology | Function and homology information guanyl-nucleotide exchange factor complex / negative regulation of cytoplasmic translational initiation / guanosine tetraphosphate binding / stringent response / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / translational elongation / Group I intron splicing / RNA folding ...guanyl-nucleotide exchange factor complex / negative regulation of cytoplasmic translational initiation / guanosine tetraphosphate binding / stringent response / ornithine decarboxylase inhibitor activity / transcription antitermination factor activity, RNA binding / misfolded RNA binding / translational elongation / Group I intron splicing / RNA folding / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis / translation elongation factor activity / negative regulation of cytoplasmic translation / translational termination / DnaA-L2 complex / four-way junction DNA binding / translation repressor activity / negative regulation of translational initiation / negative regulation of DNA-templated DNA replication initiation / regulation of mRNA stability / mRNA regulatory element binding translation repressor activity / ribosome assembly / assembly of large subunit precursor of preribosome / positive regulation of RNA splicing / cytosolic ribosome assembly / transcription elongation factor complex / regulation of DNA-templated transcription elongation / DNA endonuclease activity / response to reactive oxygen species / transcription antitermination / regulation of cell growth / translational initiation / DNA-templated transcription termination / maintenance of translational fidelity / response to radiation / mRNA 5'-UTR binding / ribosomal small subunit biogenesis / small ribosomal subunit rRNA binding / large ribosomal subunit / ribosome biogenesis / ribosome binding / regulation of translation / ribosomal small subunit assembly / large ribosomal subunit rRNA binding / 5S rRNA binding / transferase activity / small ribosomal subunit / cytosolic small ribosomal subunit / ribosomal large subunit assembly / cytoplasmic translation / cytosolic large ribosomal subunit / tRNA binding / molecular adaptor activity / 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 / DNA binding / RNA binding / zinc ion binding / membrane / plasma membrane / cytosol / cytoplasm Similarity search - Function | |||||||||
Biological species | Escherichia coli (E. coli) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å | |||||||||
Authors | Fislage, M. / Brown, Z. / Frank, J. | |||||||||
Funding support | United States, 2items
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Citation | Journal: Nucleic Acids Res / Year: 2018 Title: Cryo-EM shows stages of initial codon selection on the ribosome by aa-tRNA in ternary complex with GTP and the GTPase-deficient EF-TuH84A. Authors: Marcus Fislage / Jingji Zhang / Zuben Patrick Brown / Chandra Sekhar Mandava / Suparna Sanyal / Måns Ehrenberg / Joachim Frank / Abstract: The GTPase EF-Tu in ternary complex with GTP and aminoacyl-tRNA (aa-tRNA) promotes rapid and accurate delivery of cognate aa-tRNAs to the ribosomal A site. Here we used cryo-EM to study the molecular ...The GTPase EF-Tu in ternary complex with GTP and aminoacyl-tRNA (aa-tRNA) promotes rapid and accurate delivery of cognate aa-tRNAs to the ribosomal A site. Here we used cryo-EM to study the molecular origins of the accuracy of ribosome-aided recognition of a cognate ternary complex and the accuracy-amplifying role of the monitoring bases A1492, A1493 and G530 of the 16S rRNA. We used the GTPase-deficient EF-Tu variant H84A with native GTP, rather than non-cleavable GTP analogues, to trap a near-cognate ternary complex in high-resolution ribosomal complexes of varying codon-recognition accuracy. We found that ribosome complexes trapped by GTPase-deficicent ternary complex due to the presence of EF-TuH84A or non-cleavable GTP analogues have very similar structures. We further discuss speed and accuracy of initial aa-tRNA selection in terms of conformational changes of aa-tRNA and stepwise activation of the monitoring bases at the decoding center of the ribosome. | |||||||||
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 | 5wdt.cif.gz | 3.9 MB | Display | PDBx/mmCIF format |
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PDB format | pdb5wdt.ent.gz | Display | PDB format | |
PDBx/mmJSON format | 5wdt.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 5wdt_validation.pdf.gz | 1.8 MB | Display | wwPDB validaton report |
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Full document | 5wdt_full_validation.pdf.gz | 1.9 MB | Display | |
Data in XML | 5wdt_validation.xml.gz | 246.3 KB | Display | |
Data in CIF | 5wdt_validation.cif.gz | 425.1 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/wd/5wdt ftp://data.pdbj.org/pub/pdb/validation_reports/wd/5wdt | HTTPS FTP |
-Related structure data
Related structure data | 8813MC 8814C 8815C 8826C 8828C 8829C 5we4C 5we6C 5wf0C 5wfkC 5wfsC 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 , 6 types, 7 molecules ABavwxy
#1: RNA chain | Mass: 941548.438 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 / References: GenBank: 731469900 | ||||
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#2: RNA chain | Mass: 38813.133 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 / References: GenBank: 1174070234 | ||||
#34: RNA chain | Mass: 499239.000 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 / References: GenBank: 1114169151 | ||||
#55: RNA chain | Mass: 24818.893 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 / References: GenBank: 731469900 #56: RNA chain | | Mass: 3773.280 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: in vitro transcription vector pT7-TP(deltai) / Source: (synth.) Escherichia coli (E. coli) #57: RNA chain | | Mass: 24649.912 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 |
+50S ribosomal protein ... , 31 types, 31 molecules CDEFGHIJKLMNOPQRSTUVWXYZ0123456
-30S ribosomal protein ... , 20 types, 20 molecules bcdefghijklmnopqrstu
#35: Protein | Mass: 24253.943 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 / References: UniProt: P0A7V0 |
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#36: Protein | Mass: 23078.785 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 / References: UniProt: P0A7V3 |
#37: Protein | Mass: 23383.002 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 / References: UniProt: P0A7V8 |
#38: Protein | Mass: 16532.088 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 / References: UniProt: P0A7W1 |
#39: Protein | Mass: 11669.371 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 / References: UniProt: P02358 |
#40: Protein | Mass: 16861.523 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 / References: UniProt: P02359 |
#41: Protein | Mass: 14015.361 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 / References: UniProt: P0A7W7 |
#42: Protein | Mass: 14554.882 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 / References: UniProt: P0A7X3 |
#43: Protein | Mass: 11196.988 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 / References: UniProt: P0A7R5 |
#44: Protein | Mass: 12388.068 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 / References: UniProt: P0A7R9 |
#45: Protein | Mass: 13436.703 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 / References: UniProt: P0A7S3 |
#46: Protein | Mass: 12738.911 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 / References: UniProt: P0A7S9 |
#47: Protein | Mass: 11546.442 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 / References: UniProt: P0AG59 |
#48: Protein | Mass: 10159.621 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 / References: UniProt: P0ADZ4 |
#49: Protein | Mass: 9207.572 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 / References: UniProt: P0A7T3 |
#50: Protein | Mass: 9263.946 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 / References: UniProt: P0AG63 |
#51: Protein | Mass: 7606.768 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 / References: UniProt: P0A7T7 |
#52: Protein | Mass: 9057.626 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 / References: UniProt: P0A7U3 |
#53: Protein | Mass: 9506.190 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 / References: UniProt: P0A7U7 |
#54: Protein | Mass: 7763.073 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 / References: UniProt: P68679 |
-Protein , 1 types, 1 molecules z
#58: Protein | Mass: 43172.230 Da / Num. of mol.: 1 / Mutation: H84A Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Strain: MRE 600 / Gene: tufB, b3980, JW3943 / Production host: Escherichia coli (E. coli) / References: UniProt: P0CE48 |
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-Non-polymers , 6 types, 2832 molecules
#59: Chemical | ChemComp-FME / | ||||||||
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#60: Chemical | ChemComp-MG / #61: Chemical | ChemComp-K / #62: Chemical | ChemComp-PHE / | #63: Chemical | ChemComp-GNP / | #64: Water | ChemComp-HOH / | |
-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: 70S ribosome-EF-Tu H84A complex with GppNHp / Type: RIBOSOME / Entity ID: #1-#58 / Source: MULTIPLE SOURCES | |||||||||||||||||||||||||||||||||||
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Source (natural) | Organism: Escherichia coli (E. coli) | |||||||||||||||||||||||||||||||||||
Buffer solution | pH: 7.5 | |||||||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 300 nM 70S ribosome in final mixture | |||||||||||||||||||||||||||||||||||
Specimen support | Details: grids were manually coated with gold and the carbon support was removed by glow discharge. gas mixture: 20% hydrogen/80% oxygen power: 15 W Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 | |||||||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K / Details: 5 second blot time, no wait time |
-Electron microscopy imaging
Experimental equipment | Model: Tecnai Polara / Image courtesy: FEI Company |
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Microscopy | Model: FEI POLARA 300 |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 39000 X / Calibrated magnification: 51020 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1500 nm / Calibrated defocus min: 500 nm / Calibrated defocus max: 4000 nm / Cs: 2.26 mm / C2 aperture diameter: 30 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: OTHER |
Image recording | Average exposure time: 0.2 sec. / Electron dose: 67 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1735 |
Image scans | Sampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 40 / Used frames/image: 1-40 |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 134718 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 55276 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 97 / Protocol: OTHER / Space: RECIPROCAL |