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- PDB-5zzm: E. coli 50S subunit bound HflX protein in presence of ATP (AMP-PN... -

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Basic information

Entry
Database: PDB / ID: 5zzm
TitleE. coli 50S subunit bound HflX protein in presence of ATP (AMP-PNP) and GTP (GMP-PNP) analogs.
Components
  • 23S rRNA
  • 5S rRNA
  • GTPase HflX
KeywordsRIBOSOME / ATPase / RNA helicase / Heat stress
Function / homology
Function and homology information


ribosome disassembly / guanosine tetraphosphate binding / ribosomal large subunit binding / rescue of stalled ribosome / ribosome binding / response to heat / rRNA binding / GTPase activity / GTP binding / ATP hydrolysis activity ...ribosome disassembly / guanosine tetraphosphate binding / ribosomal large subunit binding / rescue of stalled ribosome / ribosome binding / response to heat / rRNA binding / GTPase activity / GTP binding / ATP hydrolysis activity / ATP binding / metal ion binding / cytoplasm / cytosol
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 / GTP binding domain / EF-G domain III/V-like / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
: / : / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / GTPase HflX
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 8.1 Å
AuthorsDey, S.
CitationJournal: J Cell Biol / Year: 2018
Title: The universally conserved GTPase HflX is an RNA helicase that restores heat-damaged ribosomes.
Authors: Sandip Dey / Chiranjit Biswas / Jayati Sengupta /
Abstract: The ribosome-associated GTPase HflX acts as an antiassociation factor upon binding to the 50S ribosomal subunit during heat stress in Although HflX is recognized as a guanosine triphosphatase, ...The ribosome-associated GTPase HflX acts as an antiassociation factor upon binding to the 50S ribosomal subunit during heat stress in Although HflX is recognized as a guanosine triphosphatase, several studies have shown that the N-terminal domain 1 of HflX is capable of hydrolyzing adenosine triphosphate (ATP), but the functional role of its adenosine triphosphatase (ATPase) activity remains unknown. We demonstrate that HflX possesses ATP-dependent RNA helicase activity and is capable of unwinding large subunit ribosomal RNA. A cryo-electron microscopy structure of the 50S-HflX complex in the presence of nonhydrolyzable analogues of ATP and guanosine triphosphate hints at a mode of action for the RNA helicase and suggests the linker helical domain may have a determinant role in RNA unwinding. Heat stress results in inactivation of the ribosome, and we show that HflX can restore heat-damaged ribosomes and improve cell survival.
History
DepositionJun 3, 2018Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 27, 2018Provider: repository / Type: Initial release
Revision 1.1Sep 5, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_volume ..._citation.journal_abbrev / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: GTPase HflX
M: 5S rRNA
N: 23S rRNA


Theoretical massNumber of molelcules
Total (without water)1,028,4893
Polymers1,028,4893
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein GTPase HflX / GTP-binding protein HflX / Coordinate model: Cα atoms only


Mass: 48392.988 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: hflX, b4173, JW4131 / Plasmid: pET28a / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): BL21 / References: UniProt: P25519
#2: RNA chain 5S rRNA / Coordinate model: P atoms only


Mass: 38790.090 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 / References: GenBank: 1370526514
#3: RNA chain 23S rRNA / Coordinate model: P atoms only


Mass: 941306.188 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 / References: GenBank: 1036415628

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: E. coli 50S bound HflX protein in presence of ATP (AMP-PNP) and GTP (GMP-PNP) analogs.
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Source (natural)Organism: Escherichia coli (E. coli) / Strain: BL21
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in.
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 K

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN 910 MULTI-SPECIMEN SINGLE TILT CRYO TRANSFER HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 20 e/Å2 / Film or detector model: FEI EAGLE (4k x 4k) / Num. of grids imaged: 4

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Processing

EM software
IDNameVersionCategory
1SPIDER23.03particle selection
4EMAN22.2 finalCTF correction
7PyMOL1.7.xmodel fitting
9MDFFmodel refinement
10SPIDER23.03initial Euler assignment
13EMAN22.2 final3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 8.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 61557 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT

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