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- EMDB-3133: Cryo-EM structures of the 50S ribosome subunit bound with HflX -

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Basic information

Entry
Database: EMDB / ID: EMD-3133
TitleCryo-EM structures of the 50S ribosome subunit bound with HflX
Map dataCryo-EM structures of the 50S ribosome subunit bound with HflX
Sample
  • Sample: 50S-HflX complex
  • Complex: prokaryotic 50S ribosome subunit
  • Protein or peptide: HflX
KeywordsRibosome rescue
Function / homology
Function and homology information


ribosome disassembly / 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 ...ribosome disassembly / 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 / rescue of stalled ribosome / ribosome assembly / mRNA regulatory element binding translation repressor activity / response to reactive oxygen species / assembly of large subunit precursor of preribosome / cytosolic ribosome assembly / regulation of cell growth / DNA-templated transcription termination / : / response to radiation / mRNA 5'-UTR binding / large ribosomal subunit rRNA binding / ribosome binding / large ribosomal subunit / 5S rRNA binding / ribosomal large subunit assembly / cytosolic large ribosomal subunit / cytoplasmic translation / response to heat / 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 / ATP hydrolysis activity / DNA binding / RNA binding / zinc ion binding / ATP binding / metal ion binding / cytosol / cytoplasm
Similarity search - Function
HflX, C-terminal domain / HflX C-terminal domain / GTPase HflX / GTPase HflX, N-terminal / HflX-type guanine nucleotide-binding (G) domain / GTP-binding protein, middle domain / GTPase HflX, N-terminal domain superfamily / GTP-binding GTPase N-terminal / GTP-binding GTPase Middle Region / HflX-type guanine nucleotide-binding (G) domain profile. ...HflX, C-terminal domain / HflX C-terminal domain / GTPase HflX / GTPase HflX, N-terminal / HflX-type guanine nucleotide-binding (G) domain / GTP-binding protein, middle domain / GTPase HflX, N-terminal domain superfamily / GTP-binding GTPase N-terminal / GTP-binding GTPase Middle Region / HflX-type guanine nucleotide-binding (G) domain profile. / 50S ribosome-binding GTPase / Ribosomal protein L1, bacterial-type / GTP binding domain / Ribosomal protein L10, eubacterial, conserved site / Ribosomal protein L10 signature. / Ribosomal protein L10 / EF-G domain III/V-like / : / Ribosomal protein L1, conserved site / Ribosomal protein L1 signature. / Ribosomal protein L1 / Ribosomal protein L1, 3-layer alpha/beta-sandwich / Ribosomal protein L25, short-form / Ribosomal protein L11, bacterial-type / Ribosomal protein L1-like / Ribosomal protein L1/ribosomal biogenesis protein / Ribosomal protein L1p/L10e family / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / Ribosomal protein L11, conserved site / Ribosomal protein L10-like domain superfamily / Ribosomal protein L11 signature. / Ribosomal protein L16 signature 1. / : / Ribosomal protein L10 / Ribosomal protein L10P / 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
Similarity search - Domain/homology
Large ribosomal subunit protein uL15 / Large ribosomal subunit protein uL10 / 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 uL15 / Large ribosomal subunit protein uL10 / 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 HflX / 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) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsZhang Y / Mandava CS / Cao W / Li X / Zhang D / Li N / Zhang X / Qin Y / Mi K / Lei J ...Zhang Y / Mandava CS / Cao W / Li X / Zhang D / Li N / Zhang X / Qin Y / Mi K / Lei J / Sanyal S / Gao N
CitationJournal: Nat Struct Mol Biol / Year: 2015
Title: HflX is a ribosome-splitting factor rescuing stalled ribosomes under stress conditions.
Authors: Yanqing Zhang / Chandra Sekhar Mandava / Wei Cao / Xiaojing Li / Dejiu Zhang / Ningning Li / Yixiao Zhang / Xiaoxiao Zhang / Yan Qin / Kaixia Mi / Jianlin Lei / Suparna Sanyal / Ning Gao /
Abstract: Adverse cellular conditions often lead to nonproductive translational stalling and arrest of ribosomes on mRNAs. Here, we used fast kinetics and cryo-EM to characterize Escherichia coli HflX, a ...Adverse cellular conditions often lead to nonproductive translational stalling and arrest of ribosomes on mRNAs. Here, we used fast kinetics and cryo-EM to characterize Escherichia coli HflX, a GTPase with unknown function. Our data reveal that HflX is a heat shock-induced ribosome-splitting factor capable of dissociating vacant as well as mRNA-associated ribosomes with deacylated tRNA in the peptidyl site. Structural data demonstrate that the N-terminal effector domain of HflX binds to the peptidyl transferase center in a strikingly similar manner as that of the class I release factors and induces dramatic conformational changes in central intersubunit bridges, thus promoting subunit dissociation. Accordingly, loss of HflX results in an increase in stalled ribosomes upon heat shock. These results suggest a primary role of HflX in rescuing translationally arrested ribosomes under stress conditions.
History
DepositionAug 23, 2015-
Header (metadata) releaseSep 16, 2015-
Map releaseOct 14, 2015-
UpdateDec 2, 2015-
Current statusDec 2, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.4
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 1.4
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5ady
  • Surface level: 1.4
  • 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_3133.map.gz / Format: CCP4 / Size: 100.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structures of the 50S ribosome subunit bound with HflX
Voxel sizeX=Y=Z: 1.1659 Å
Density
Contour LevelBy AUTHOR: 1.4 / Movie #1: 1.4
Minimum - Maximum-2.33597445 - 5.85036802
Average (Standard dev.)0.04000991 (±0.41606173)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-149-149-149
Dimensions300300300
Spacing300300300
CellA=B=C: 349.77 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.16591.16591.1659
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z349.770349.770349.770
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-149-149-149
NC/NR/NS300300300
D min/max/mean-2.3365.8500.040

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Supplemental data

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Sample components

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Entire : 50S-HflX complex

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

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Supramolecule #1000: 50S-HflX complex

SupramoleculeName: 50S-HflX 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) / Location in cell: Cytoplasm
Molecular weightExperimental: 1.5 MDa / Theoretical: 1.5 MDa

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Macromolecule #1: HflX

MacromoleculeName: HflX / type: protein_or_peptide / ID: 1
Details: GMPPNP was bound with HflX in the 50S-HflX complex.
Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Location in cell: Cytoplasm
Molecular weightExperimental: 50 KDa / Theoretical: 50 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria) / Recombinant plasmid: pET28a
SequenceUniProtKB: GTPase HflX

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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: 2.70 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DateJan 7, 2012
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Number real images: 6022 / 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: 4.5 Å / Resolution method: OTHER / Software - Name: Spider, Relion / Number images used: 384206

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Atomic model buiding 1

Initial modelPDB ID:

3fik
PDB Unreleased entry

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

PDB-5ady:
Cryo-EM structures of the 50S ribosome subunit bound with HflX

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Atomic model buiding 2

Initial modelPDB ID:
SoftwareName: MDFF
DetailsAtomic model of E. coli HflX was modeled from the crystal structure of Sulfolobus solfataricus HflX (PDB id: 3KXI).The homology modeling was performed with MODELLER. The model of the CTD of E. coli HflX was independently modeled by I-TASSER (template PDB code: 2WBM, residues 164-232). The switch I region disordered in the crystal structure of S. solfataricus HflX was modeled using the crystal structure of S. thermophilus NFeoB (PDB id: 3B1X) as a template. GMPPNP was derived from a previous model (PDB id: 3B1X) and docked into the atomic model of the E. coli HflX.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-5ady:
Cryo-EM structures of the 50S ribosome subunit bound with HflX

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