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- EMDB-2605: Cryo-EM structures of the 50S ribosome subunit bound with ObgE -

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Basic information

Entry
Database: EMDB / ID: EMD-2605
TitleCryo-EM structures of the 50S ribosome subunit bound with ObgE
Map dataReconstruction of 50S-ObgE complex
Sample
  • Sample: 50S-ObgE complex
  • Complex: prokaryotic 50S ribosome subunit
  • Protein or peptide: ObgE
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 / negative regulation of translational initiation / translation repressor activity / negative regulation of DNA-templated DNA replication initiation / ribosome assembly / mRNA regulatory element binding translation repressor activity / response to reactive oxygen species / assembly of large subunit precursor of preribosome / cytosolic ribosome assembly / chromosome segregation / regulation of cell growth / DNA-templated transcription termination / : / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / response to radiation / mRNA 5'-UTR binding / GDP binding / large ribosomal subunit rRNA binding / ribosome binding / ribosomal large subunit assembly / large ribosomal subunit / 5S rRNA binding / cytosolic large ribosomal subunit / cytoplasmic translation / transferase activity / tRNA binding / negative regulation of translation / ribosome / rRNA binding / structural constituent of ribosome / translation / response to antibiotic / GTPase activity / mRNA binding / negative regulation of DNA-templated transcription / GTP binding / magnesium ion binding / DNA binding / RNA binding / zinc ion binding / cytosol / cytoplasm
Similarity search - Function
GTP1/OBG, conserved site / GTP1/OBG family signature. / OBG-type GTPase / GTP1/OBG domain / GTP-binding protein Obg/CgtA / GTP1/OBG domain superfamily / GTP1/OBG / Obg domain profile. / OBG-type guanine nucleotide-binding (G) domain / OBG-type guanine nucleotide-binding (G) domain profile. ...GTP1/OBG, conserved site / GTP1/OBG family signature. / OBG-type GTPase / GTP1/OBG domain / GTP-binding protein Obg/CgtA / GTP1/OBG domain superfamily / GTP1/OBG / Obg domain profile. / OBG-type guanine nucleotide-binding (G) domain / OBG-type guanine nucleotide-binding (G) domain profile. / 50S ribosome-binding GTPase / Ribosomal protein L1, bacterial-type / GTP binding domain / Ribosomal protein L1, conserved site / Ribosomal protein L1 / Ribosomal protein L1 signature. / Ribosomal protein L1, 3-layer alpha/beta-sandwich / Ribosomal protein L25, short-form / Ribosomal protein L1-like / Ribosomal protein L1/ribosomal biogenesis protein / Ribosomal protein L1p/L10e family / Ribosomal protein L11, bacterial-type / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / Ribosomal protein L11, conserved site / Ribosomal protein L11 signature. / Ribosomal protein L16 signature 1. / : / Ribosomal protein L6, conserved site / Ribosomal protein L6 signature 1. / Ribosomal protein L16, conserved site / Ribosomal protein L16 signature 2. / Ribosomal protein L17 signature. / Ribosomal protein L9 signature. / Ribosomal protein L9, bacteria/chloroplast / Ribosomal protein L9, C-terminal / Ribosomal protein L9, C-terminal domain / Ribosomal protein L9, C-terminal domain superfamily / Ribosomal L25p family / Ribosomal protein L25 / Ribosomal protein L11, N-terminal / Ribosomal protein L11, N-terminal domain / Ribosomal protein L11/L12 / Ribosomal protein L11, C-terminal / Ribosomal protein L11, C-terminal domain superfamily / Ribosomal protein L11/L12, N-terminal domain superfamily / Ribosomal protein L11, RNA binding domain / Ribosomal protein L11/L12 / Ribosomal protein L28/L24 superfamily / Ribosomal protein L36 signature. / Ribosomal protein L25/Gln-tRNA synthetase, N-terminal / Ribosomal protein L32p, bacterial type / Ribosomal protein L25/Gln-tRNA synthetase, anti-codon-binding domain superfamily / Ribosomal protein L9, N-terminal domain superfamily / Ribosomal protein L9 / Ribosomal protein L9, N-terminal / Ribosomal protein L9, N-terminal domain / Ribosomal protein L28 / Ribosomal protein L35, conserved site / Ribosomal protein L35 signature. / Ribosomal protein L33, conserved site / Ribosomal protein L33 signature. / Ribosomal protein L35, non-mitochondrial / Ribosomal protein L5, bacterial-type / Ribosomal protein L6, bacterial-type / Ribosomal protein L18, bacterial-type / Ribosomal protein L19, conserved site / Ribosomal protein L19 signature. / Ribosomal protein L36 / Ribosomal protein L36 superfamily / Ribosomal protein L36 / Ribosomal protein L9/RNase H1, N-terminal / Ribosomal protein L20 signature. / Ribosomal protein L27, conserved site / Ribosomal protein L27 signature. / Ribosomal protein L14P, bacterial-type / Ribosomal protein L34, conserved site / Ribosomal protein L34 signature. / Ribosomal protein L22, bacterial/chloroplast-type / Ribosomal protein L35 / Ribosomal protein L35 superfamily / Ribosomal protein L2, bacterial/organellar-type / Ribosomal protein L35 / Ribosomal L28 family / Ribosomal protein L33 / Ribosomal protein L33 / Ribosomal protein L28/L24 / Ribosomal protein L33 superfamily / : / Ribosomal protein L30, bacterial-type / Ribosomal protein L16 / Ribosomal protein L18 / Ribosomal L18 of archaea, bacteria, mitoch. and chloroplast / L28p-like / Ribosomal protein L20 / Ribosomal protein L20 / Ribosomal protein L20, C-terminal / Ribosomal protein L21 / Ribosomal protein L27 / Ribosomal L27 protein
Similarity search - Domain/homology
Large ribosomal subunit protein uL15 / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein uL1 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL28 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein bL32 / Large ribosomal subunit protein bL33 ...Large ribosomal subunit protein uL15 / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein uL1 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL28 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein bL32 / Large ribosomal subunit protein bL33 / Large ribosomal subunit protein bL34 / Large ribosomal subunit protein bL35 / Large ribosomal subunit protein bL36A / Large ribosomal subunit protein bL9 / Large ribosomal subunit protein uL13 / Large ribosomal subunit protein uL14 / Large ribosomal subunit protein uL16 / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein bL17 / Large ribosomal subunit protein bL21 / Large ribosomal subunit protein uL30 / Large ribosomal subunit protein uL6 / Large ribosomal subunit protein uL18 / GTPase ObgE/CgtA / Large ribosomal subunit protein uL2 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein uL24 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL22 / Large ribosomal subunit protein uL5 / Large ribosomal subunit protein bL25
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.5 Å
AuthorsFeng 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
CitationJournal: 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
DepositionMar 6, 2014-
Header (metadata) releaseMar 19, 2014-
Map releaseJun 4, 2014-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.045
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4csu
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_2605.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of 50S-ObgE complex
Voxel sizeX=Y=Z: 1.5 Å
Density
Contour LevelBy AUTHOR: 0.045 / Movie #1: 0.045
Minimum - Maximum-0.04568339 - 0.19051541
Average (Standard dev.)0.00172401 (±0.016385)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 384.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.51.51.5
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z384.000384.000384.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0460.1910.002

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Supplemental data

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Sample components

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Entire : 50S-ObgE complex

EntireName: 50S-ObgE complex
Components
  • Sample: 50S-ObgE complex
  • Complex: prokaryotic 50S ribosome subunit
  • Protein or peptide: ObgE

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Supramolecule #1000: 50S-ObgE complex

SupramoleculeName: 50S-ObgE complex / type: sample / ID: 1000 / Number unique components: 2
Molecular weightExperimental: 1.5 MDa / Theoretical: 1.5 MDa

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Supramolecule #1: prokaryotic 50S ribosome subunit

SupramoleculeName: prokaryotic 50S ribosome subunit / type: complex / ID: 1 / Name.synonym: 50S subunit / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: LSU 50S
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K12 / Location in cell: cytoplasm
Molecular weightExperimental: 1.5 MDa / Theoretical: 1.5 MDa

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Macromolecule #1: ObgE

MacromoleculeName: ObgE / type: protein_or_peptide / ID: 1 / Name.synonym: CgtAE / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K12
Molecular weightExperimental: 50 KDa / Theoretical: 43 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria) / Recombinant strain: BL21 / Recombinant plasmid: pET28a

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5 / Details: 20mM Tris-HCl, 100mM NH4Cl, 10mM MgCl2
GridDetails: 200 mesh copper grid with thin carbon support, glow discharged
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV / Method: Blot for 1 seconds before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 59000
Sample stageSpecimen holder: Liquid Nitrogen cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DateJul 27, 2011
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Average electron dose: 20 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionApplied 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
DetailsParticles 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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Atomic model buiding 1

Initial modelPDB ID:

3ofc
PDB Unreleased entry

SoftwareName: MDFF
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-4csu:
Cryo-EM structures of the 50S ribosome subunit bound with ObgE

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Atomic model buiding 2

Initial modelPDB ID:
SoftwareName: MDFF
DetailsThe 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)
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-4csu:
Cryo-EM structures of the 50S ribosome subunit bound with ObgE

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