|Entry||Database: PDB / ID: 5zzm|
|Title||E. coli 50S subunit bound HflX protein in presence of ATP (AMP-PNP) and GTP (GMP-PNP) analogs.|
|Keywords||RIBOSOME / ATPase / RNA helicase / Heat stress|
|Function / homology||GTP binding domain / GTP-binding protein, middle domain / P-loop containing nucleoside triphosphate hydrolase / GTPase HflX, N-terminal / GTPase HflX / EF-G domain III/V-like / 50S ribosome-binding GTPase / HflX-type guanine nucleotide-binding (G) domain / GTP-binding GTPase Middle Region / GTP-binding GTPase N-terminal ...GTP binding domain / GTP-binding protein, middle domain / P-loop containing nucleoside triphosphate hydrolase / GTPase HflX, N-terminal / GTPase HflX / EF-G domain III/V-like / 50S ribosome-binding GTPase / HflX-type guanine nucleotide-binding (G) domain / GTP-binding GTPase Middle Region / GTP-binding GTPase N-terminal / HflX-type guanine nucleotide-binding (G) domain profile. / ribosome disassembly / rescue of stalled ribosome / peptidyl-serine autophosphorylation / ribosomal large subunit binding / ribosome binding / response to heat / rRNA binding / ATPase activity / GTPase activity / GTP binding / ATP binding / metal ion binding / cytosol / cytoplasm / GTPase HflX / gb:1370526514: / gb:1036415628: |
Function and homology information
|Specimen source||Escherichia coli (E. coli)|
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 8.1 Å resolution|
|Citation||Journal: 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.
SummaryFull reportAbout validation report
|Date||Deposition: Jun 3, 2018 / Release: Jun 27, 2018|
|Structure viewer||Molecule: |
Downloads & links
A: GTPase HflX
M: 5S rRNA
N: 23S rRNA
|#1: Protein/peptide|| |
Mass: 48392.988 Da / Num. of mol.: 1
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: hflX, b4173, JW4131 / Plasmid name: pET28a / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): BL21 / References: UniProt: P25519
|#2: RNA chain|| |
Mass: 38790.090 Da / Num. of mol.: 1 / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 / References: GenBank: 1370526514
|#3: RNA chain|| |
Mass: 941306.188 Da / Num. of mol.: 1 / Source: (natural) Escherichia coli (E. coli) / Strain: MRE 600 / References: GenBank: 1036415628
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / Reconstruction method: single particle reconstruction|
|Component||Name: E. coli 50S bound HflX protein in presence of ATP (AMP-PNP) and GTP (GMP-PNP) analogs.|
Type: COMPLEX / Entity ID: 1,
|Source (natural)||Organism: Escherichia coli (E. coli) / Strain: BL21||Buffer solution||pH: 7.5||Specimen||Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES||Specimen support||Grid material: COPPER / Grid mesh size: 300||Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 kelvins|
-Electron microscopy imaging
Model: Tecnai Polara / Image courtesy: FEI Company
|Microscopy||Microscope model: FEI POLARA 300|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: DIFFRACTION|
|Specimen holder||Cryogen: NITROGEN|
Specimen holder model: GATAN 910 MULTI-SPECIMEN SINGLE TILT CRYO TRANSFER HOLDER
|Image recording||Average exposure time: 2 sec. / Electron dose: 20 e/Å2 / Film or detector model: FEI EAGLE (4k x 4k) / Number of grids imaged: 4|
|CTF correction||Type: PHASE FLIPPING ONLY|
|3D reconstruction||Resolution: 8.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 61557 / Symmetry type: POINT|
|Atomic model building||Ref protocol: FLEXIBLE FIT|
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