+Open data
-Basic information
Entry | Database: PDB / ID: 7bv8 | |||||||||||||||
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Title | Mature 50S ribosomal subunit from RrmJ knock out E.coli strain | |||||||||||||||
Components |
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Keywords | RIBOSOME / ribosome assembly / ribosome biogenesis | |||||||||||||||
Function / homology | Function and homology information stringent response / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis / negative regulation of cytoplasmic translation / translational termination / DnaA-L2 complex / translation repressor activity / negative regulation of DNA-templated DNA replication initiation ...stringent response / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis / negative regulation of cytoplasmic translation / translational termination / DnaA-L2 complex / translation repressor activity / negative regulation of DNA-templated DNA replication initiation / ribosome assembly / mRNA regulatory element binding translation repressor activity / assembly of large subunit precursor of preribosome / cytosolic ribosome assembly / response to reactive oxygen species / regulation of cell growth / DNA-templated transcription termination / response to radiation / ribosomal large subunit assembly / mRNA 5'-UTR binding / large ribosomal subunit / ribosome binding / transferase activity / 5S rRNA binding / large ribosomal subunit rRNA binding / cytosolic large ribosomal subunit / tRNA binding / cytoplasmic translation / rRNA binding / negative regulation of translation / ribosome / structural constituent of ribosome / translation / response to antibiotic / negative regulation of DNA-templated transcription / mRNA binding / DNA binding / RNA binding / zinc ion binding / cytoplasm / cytosol Similarity search - Function | |||||||||||||||
Biological species | Escherichia coli (E. coli) | |||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.14 Å | |||||||||||||||
Authors | Wang, W. / Li, W.Q. / Ge, X.L. / Yan, K.G. / Mandava, C.S. / Sanyal, S. / Gao, N. | |||||||||||||||
Funding support | China, 4items
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Citation | Journal: Proc Natl Acad Sci U S A / Year: 2020 Title: Loss of a single methylation in 23S rRNA delays 50S assembly at multiple late stages and impairs translation initiation and elongation. Authors: Wei Wang / Wanqiu Li / Xueliang Ge / Kaige Yan / Chandra Sekhar Mandava / Suparna Sanyal / Ning Gao / Abstract: Ribosome biogenesis is a complex process, and dozens of factors are required to facilitate and regulate the subunit assembly in bacteria. The 2'-O-methylation of U2552 in 23S rRNA by ...Ribosome biogenesis is a complex process, and dozens of factors are required to facilitate and regulate the subunit assembly in bacteria. The 2'-O-methylation of U2552 in 23S rRNA by methyltransferase RrmJ is a crucial step in late-stage assembly of the 50S subunit. Its absence results in severe growth defect and marked accumulation of pre50S assembly intermediates. In the present work, we employed cryoelectron microscopy to characterize a set of late-stage pre50S particles isolated from an Δ strain. These assembly intermediates (solved at 3.2 to 3.8 Å resolution) define a collection of late-stage particles on a progressive assembly pathway. Apart from the absence of L16, L35, and L36, major structural differences between these intermediates and the mature 50S subunit are clustered near the peptidyl transferase center, such as H38, H68-71, and H89-93. In addition, the ribosomal A-loop of the mature 50S subunit from Δ strain displays large local flexibility on nucleotides next to unmethylated U2552. Fast kinetics-based biochemical assays demonstrate that the Δ 50S subunit is only 50% active and two times slower than the WT 50S subunit in rapid subunit association. While the Δ 70S ribosomes show no defect in peptide bond formation, peptide release, and ribosome recycling, they translocate with 20% slower rate than the WT ribosomes in each round of elongation. These defects amplify during synthesis of the full-length proteins and cause overall defect in protein synthesis. In conclusion, our data reveal the molecular roles of U2552 methylation in both ribosome biogenesis and protein translation. | |||||||||||||||
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 | 7bv8.cif.gz | 2 MB | Display | PDBx/mmCIF format |
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PDB format | pdb7bv8.ent.gz | 1.5 MB | Display | PDB format |
PDBx/mmJSON format | 7bv8.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7bv8_validation.pdf.gz | 1.2 MB | Display | wwPDB validaton report |
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Full document | 7bv8_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | 7bv8_validation.xml.gz | 120.1 KB | Display | |
Data in CIF | 7bv8_validation.cif.gz | 215.9 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/bv/7bv8 ftp://data.pdbj.org/pub/pdb/validation_reports/bv/7bv8 | HTTPS FTP |
-Related structure data
Related structure data | 30215MC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-RNA chain , 2 types, 2 molecules AB
#1: RNA chain | Mass: 941305.188 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (strain K12) (bacteria) / Strain: K12 |
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#2: RNA chain | Mass: 38790.090 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (strain K12) (bacteria) / Strain: K12 |
+50S ribosomal protein ... , 30 types, 30 molecules CDEFGHIJKLMNOPQRSTUVWXYZabcdef
-Details
Source details | The E.coli. stain is BW25113, which is a derivative of E.coli. K12 strain. |
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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: Mature 50S ribosomal subunit from RrmJ knock out E.coli strain Type: RIBOSOME Details: Mature 50S ribosomal subunit from RrmJ knock out E.coli strain Entity ID: all / Source: NATURAL |
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Molecular weight | Value: 1.5 MDa / Experimental value: NO |
Source (natural) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7.5 Details: Solutions were made fresh form concentrated to avoid microbial contamination. |
Specimen | Conc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse. |
Specimen support | Details: The grid was coated with continuous carbon film. / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot for 2 seconds before plunging |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS / Details: Preliminary grid screening was performed manually. |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 75000 X / Calibrated magnification: 75000 X / Nominal defocus max: -2000 nm / Nominal defocus min: -1000 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: 45 e/Å2 / Film or detector model: FEI FALCON II (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1164 |
Image scans | Width: 4096 / Height: 4096 / Movie frames/image: 28 / Used frames/image: 3-28 |
-Processing
Software | Name: PHENIX / Version: 1.16_3549: / Classification: refinement | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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Image processing | Details: The images were normalized and manually selected. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CTF correction | Details: CTF amplitude correction was performed following 3D reconstruction. Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 233762 Details: Images were binned and lowpass filtered for autopicking. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.14 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 98194 / Algorithm: SIMULTANEOUS ITERATIVE (SIRT) Details: A soft mask was added to improve resolution at the final refinement. Num. of class averages: 1 / Symmetry type: POINT | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building |
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Atomic model building |
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Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 51.84 Å2 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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