+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-30215 | |||||||||||||||
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Title | Mature 50S ribosomal subunit from RrmJ knock out E.coli strain | |||||||||||||||
Map data | mature 50S from mutation strains | |||||||||||||||
Sample |
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Keywords | ribosome assembly / ribosome biogenesis / RIBOSOME | |||||||||||||||
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 / mRNA regulatory element binding translation repressor activity / ribosome assembly / assembly of large subunit precursor of preribosome / cytosolic ribosome assembly / ribosomal large subunit assembly / response to reactive oxygen species / regulation of cell growth / DNA-templated transcription termination / response to radiation / mRNA 5'-UTR binding / large ribosomal subunit / ribosome binding / transferase activity / 5S rRNA binding / large ribosomal subunit rRNA binding / cytosolic large ribosomal subunit / cytoplasmic translation / tRNA binding / 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) / Escherichia coli (strain K12) (bacteria) | |||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.14 Å | |||||||||||||||
Authors | Wang W / Li WQ | |||||||||||||||
Funding support | China, 4 items
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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 | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_30215.map.gz | 116.9 MB | EMDB map data format | |
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Header (meta data) | emd-30215-v30.xml emd-30215.xml | 50.9 KB 50.9 KB | Display Display | EMDB header |
Images | emd_30215.png | 53.3 KB | ||
Filedesc metadata | emd-30215.cif.gz | 10.9 KB | ||
Others | emd_30215_additional_1.map.gz emd_30215_additional_2.map.gz emd_30215_additional_3.map.gz | 117 MB 117.1 MB 117.1 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-30215 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-30215 | HTTPS FTP |
-Validation report
Summary document | emd_30215_validation.pdf.gz | 652.1 KB | Display | EMDB validaton report |
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Full document | emd_30215_full_validation.pdf.gz | 651.7 KB | Display | |
Data in XML | emd_30215_validation.xml.gz | 6.6 KB | Display | |
Data in CIF | emd_30215_validation.cif.gz | 7.5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-30215 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-30215 | HTTPS FTP |
-Related structure data
Related structure data | 7bv8MC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_30215.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | mature 50S from mutation strains | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.08 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Supplemental map: emd 30215 additional 1.map
File | emd_30215_additional_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Supplemental map: emd 30215 additional 2.map
File | emd_30215_additional_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Supplemental map: emd 30215 additional 3.map
File | emd_30215_additional_3.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
+Entire : Mature 50S ribosomal subunit from RrmJ knock out E.coli strain
+Supramolecule #1: Mature 50S ribosomal subunit from RrmJ knock out E.coli strain
+Macromolecule #1: 23S rRNA
+Macromolecule #2: 5S rRNA
+Macromolecule #3: 50S ribosomal protein L2
+Macromolecule #4: 50S ribosomal protein L3
+Macromolecule #5: 50S ribosomal protein L4
+Macromolecule #6: 50S ribosomal protein L5
+Macromolecule #7: 50S ribosomal protein L6
+Macromolecule #8: 50S ribosomal protein L9
+Macromolecule #9: 50S ribosomal protein L10
+Macromolecule #10: 50S ribosomal protein L11
+Macromolecule #11: 50S ribosomal protein L13
+Macromolecule #12: 50S ribosomal protein L14
+Macromolecule #13: 50S ribosomal protein L15
+Macromolecule #14: 50S ribosomal protein L16
+Macromolecule #15: 50S ribosomal protein L17
+Macromolecule #16: 50S ribosomal protein L18
+Macromolecule #17: 50S ribosomal protein L19
+Macromolecule #18: 50S ribosomal protein L20
+Macromolecule #19: 50S ribosomal protein L21
+Macromolecule #20: 50S ribosomal protein L22
+Macromolecule #21: 50S ribosomal protein L23
+Macromolecule #22: 50S ribosomal protein L24
+Macromolecule #23: 50S ribosomal protein L25
+Macromolecule #24: 50S ribosomal protein L27
+Macromolecule #25: 50S ribosomal protein L28
+Macromolecule #26: 50S ribosomal protein L29
+Macromolecule #27: 50S ribosomal protein L30
+Macromolecule #28: 50S ribosomal protein L32
+Macromolecule #29: 50S ribosomal protein L33
+Macromolecule #30: 50S ribosomal protein L34
+Macromolecule #31: 50S ribosomal protein L35
+Macromolecule #32: 50S ribosomal protein L36
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.15 mg/mL |
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Buffer | pH: 7.5 Details: Solutions were made fresh form concentrated to avoid microbial contamination. |
Grid | Model: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 7000 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.03 kPa / Details: The grid was coated with continuous carbon film. |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 2 seconds before plunging. |
Details | This sample was monodisperse. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Details | Preliminary grid screening was performed manually. |
Image recording | Film or detector model: FEI FALCON II (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Frames/image: 3-28 / Number grids imaged: 1 / Number real images: 1164 / Average electron dose: 45.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 75000 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: -2.0 µm / Nominal defocus min: -1.0 µm / Nominal magnification: 75000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Initial model |
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Refinement | Space: RECIPROCAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient | ||||||
Output model | PDB-7bv8: |