+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-2605 | |||||||||
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Title | Cryo-EM structures of the 50S ribosome subunit bound with ObgE | |||||||||
Map data | Reconstruction of 50S-ObgE complex | |||||||||
Sample |
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Keywords | (p)ppGpp / Obg / ribosome assembly / stringent response / GTPase | |||||||||
Function / homology | Function and homology information guanyl ribonucleotide binding / dormancy process / negative regulation of ribosome biogenesis / guanosine tetraphosphate binding / stringent response / ribosomal large subunit binding / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis ...guanyl ribonucleotide binding / dormancy process / negative regulation of ribosome biogenesis / guanosine tetraphosphate binding / stringent response / ribosomal large subunit binding / 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 / negative regulation of translational initiation / mRNA regulatory element binding translation repressor activity / ribosome assembly / assembly of large subunit precursor of preribosome / cytosolic ribosome assembly / response to reactive oxygen species / chromosome segregation / regulation of cell growth / DNA-templated transcription termination / response to radiation / mRNA 5'-UTR binding / GDP binding / large ribosomal subunit / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / ribosome binding / ribosomal large subunit assembly / transferase activity / large ribosomal subunit rRNA binding / 5S rRNA binding / cytoplasmic translation / cytosolic large ribosomal subunit / 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 / magnesium ion binding / DNA binding / RNA binding / zinc ion binding / cytosol / cytoplasm Similarity search - Function | |||||||||
Biological species | Escherichia coli K-12 (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 5.5 Å | |||||||||
Authors | Feng B / Mandava CS / Guo Q / Wang J / Cao W / Li N / Zhang Y / Wang Z / Wu J / Sanyal S ...Feng B / Mandava CS / Guo Q / Wang J / Cao W / Li N / Zhang Y / Wang Z / Wu J / Sanyal S / Lei J / Gao N | |||||||||
Citation | Journal: PLoS Biol / Year: 2014 Title: Structural and functional insights into the mode of action of a universally conserved Obg GTPase. Authors: Boya Feng / Chandra Sekhar Mandava / Qiang Guo / Jie Wang / Wei Cao / Ningning Li / Yixiao Zhang / Yanqing Zhang / Zhixin Wang / Jiawei Wu / Suparna Sanyal / Jianlin Lei / Ning Gao / Abstract: Obg proteins are a family of P-loop GTPases, conserved from bacteria to human. The Obg protein in Escherichia coli (ObgE) has been implicated in many diverse cellular functions, with proposed ...Obg proteins are a family of P-loop GTPases, conserved from bacteria to human. The Obg protein in Escherichia coli (ObgE) has been implicated in many diverse cellular functions, with proposed molecular roles in two global processes, ribosome assembly and stringent response. Here, using pre-steady state fast kinetics we demonstrate that ObgE is an anti-association factor, which prevents ribosomal subunit association and downstream steps in translation by binding to the 50S subunit. ObgE is a ribosome dependent GTPase; however, upon binding to guanosine tetraphosphate (ppGpp), the global regulator of stringent response, ObgE exhibits an enhanced interaction with the 50S subunit, resulting in increased equilibrium dissociation of the 70S ribosome into subunits. Furthermore, our cryo-electron microscopy (cryo-EM) structure of the 50S·ObgE·GMPPNP complex indicates that the evolutionarily conserved N-terminal domain (NTD) of ObgE is a tRNA structural mimic, with specific interactions with peptidyl-transferase center, displaying a marked resemblance to Class I release factors. These structural data might define ObgE as a specialized translation factor related to stress responses, and provide a framework towards future elucidation of functional interplay between ObgE and ribosome-associated (p)ppGpp regulators. Together with published data, our results suggest that ObgE might act as a checkpoint in final stages of the 50S subunit assembly under normal growth conditions. And more importantly, ObgE, as a (p)ppGpp effector, might also have a regulatory role in the production of the 50S subunit and its participation in translation under certain stressed conditions. Thus, our findings might have uncovered an under-recognized mechanism of translation control by environmental cues. | |||||||||
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_2605.map.gz | 59 MB | EMDB map data format | |
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Header (meta data) | emd-2605-v30.xml emd-2605.xml | 11.7 KB 11.7 KB | Display Display | EMDB header |
Images | EMD-2605.png | 119.6 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2605 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2605 | HTTPS FTP |
-Validation report
Summary document | emd_2605_validation.pdf.gz | 253.1 KB | Display | EMDB validaton report |
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Full document | emd_2605_full_validation.pdf.gz | 252.2 KB | Display | |
Data in XML | emd_2605_validation.xml.gz | 5.6 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2605 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2605 | HTTPS FTP |
-Related structure data
Related structure data | 4csuMC M: atomic model generated by this map C: citing same article (ref.) |
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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_2605.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of 50S-ObgE complex | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.5 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : 50S-ObgE complex
Entire | Name: 50S-ObgE complex |
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Components |
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-Supramolecule #1000: 50S-ObgE complex
Supramolecule | Name: 50S-ObgE complex / type: sample / ID: 1000 / Number unique components: 2 |
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Molecular weight | Experimental: 1.5 MDa / Theoretical: 1.5 MDa |
-Supramolecule #1: prokaryotic 50S ribosome subunit
Supramolecule | Name: prokaryotic 50S ribosome subunit / type: complex / ID: 1 / Name.synonym: 50S subunit / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: LSU 50S |
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Source (natural) | Organism: Escherichia coli K-12 (bacteria) / Strain: K12 / Location in cell: cytoplasm |
Molecular weight | Experimental: 1.5 MDa / Theoretical: 1.5 MDa |
-Macromolecule #1: ObgE
Macromolecule | Name: ObgE / type: protein_or_peptide / ID: 1 / Name.synonym: CgtAE / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes |
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Source (natural) | Organism: Escherichia coli K-12 (bacteria) / Strain: K12 |
Molecular weight | Experimental: 50 KDa / Theoretical: 43 KDa |
Recombinant expression | Organism: Escherichia coli BL21 (bacteria) / Recombinant strain: BL21 / Recombinant plasmid: pET28a |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.5 / Details: 20mM Tris-HCl, 100mM NH4Cl, 10mM MgCl2 |
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Grid | Details: 200 mesh copper grid with thin carbon support, glow discharged |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV / Method: Blot for 1 seconds before plunging |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Date | Jul 27, 2011 |
Image recording | Category: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Average electron dose: 20 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 59000 |
Sample stage | Specimen holder: Liquid Nitrogen cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Details | Particles were first picked using a method based on a locally normalized cross-correlation function, subjected to correspondence analysis and then manually verified. Then all particles were first classified in two groups, according to the presence or absence of ObgE on the 50S subunit using a modified supervised classification method. The resulting ObgE-containing particles were further applied to another round of 3D classification using RELION. The particles were finally split into four groups in 30 iterations using a final angle sampling of 1.8 degree. One of the four groups was used for final refinement. The refinement was performed using RELION. Amplitude correction using the B-factor sharpening approach was applied to the final volume. |
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CTF correction | Details: Each particle |
Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 5.5 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 102814 |
-Atomic model buiding 1
Initial model | PDB ID: 3ofc |
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Software | Name: MDFF |
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |
Output model | PDB-4csu: |
-Atomic model buiding 2
Initial model | PDB ID: |
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Software | Name: MDFF |
Details | The atomic model of the E. coli ObgE was built with MODELLER, using the B. subtilis and T. thermophilus Obg crystal structures (PDB IDs 1LNZ and 1UDX) |
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |
Output model | PDB-4csu: |