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- EMDB-6979: E. coli 50S subunit bound HflX protein in presence of ATP (AMP-PN... -

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Basic information

Entry
Database: EMDB / ID: 6979
TitleE. coli 50S subunit bound HflX protein in presence of ATP (AMP-PNP) and GTP (GMP-PNP) analogs.
Map dataE. coli 50S bound HflX protein in presence of ATP (AMP-PNP) and GTP (GMP-PNP) analogs.
SampleE. coli 50S bound HflX protein in presence of ATP (AMP-PNP) and GTP (GMP-PNP) analogs.:
GTPase HflX / (nucleic-acidNucleic acid) x 2
Function / homologyHflX-type guanine nucleotide-binding (G) domain / 50S ribosome-binding GTPase / HflX-type guanine nucleotide-binding (G) domain profile. / GTP-binding GTPase Middle Region / GTP-binding GTPase N-terminal / P-loop containing nucleoside triphosphate hydrolase / GTPase HflX, N-terminal / GTPase HflX / GTP-binding protein, middle domain / GTP binding domain ...HflX-type guanine nucleotide-binding (G) domain / 50S ribosome-binding GTPase / HflX-type guanine nucleotide-binding (G) domain profile. / GTP-binding GTPase Middle Region / GTP-binding GTPase N-terminal / P-loop containing nucleoside triphosphate hydrolase / GTPase HflX, N-terminal / GTPase HflX / GTP-binding protein, middle domain / GTP binding domain / EF-G domain III/V-like / ribosome disassembly / peptidyl-serine autophosphorylation / rescue of stalled ribosome / 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
Function and homology information
SourceEscherichia coli (E. coli) / Escherichia coli (strain K12) (bacteria)
Methodsingle particle reconstruction / cryo EM / 8.1 Å resolution
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.
Validation ReportPDB-ID: 5zzm

SummaryFull reportAbout validation report
DateDeposition: Jun 3, 2018 / Header (metadata) release: Jun 27, 2018 / Map release: Jun 27, 2018 / Last update: Jun 27, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-5zzm
  • Surface level: 2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_6979.map.gz (map file in CCP4 format, 37045 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
210 pix
1.89 Å/pix.
= 396.9 Å
210 pix
1.89 Å/pix.
= 396.9 Å
210 pix
1.89 Å/pix.
= 396.9 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.89 Å
Density
Contour Level:2.0 (by author), 2 (movie #1):
Minimum - Maximum-7.187189 - 15.866949
Average (Standard dev.)0.081063196 (0.78008014)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions210210210
Origin0.0.0.
Limit209.209.209.
Spacing210210210
CellA=B=C: 396.9 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.891.891.89
M x/y/z210210210
origin x/y/z0.0000.0000.000
length x/y/z396.900396.900396.900
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS210210210
D min/max/mean-7.18715.8670.081

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Supplemental data

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Sample components

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Entire E. coli 50S bound HflX protein in presence of ATP (AMP-PNP) and G...

EntireName: E. coli 50S bound HflX protein in presence of ATP (AMP-PNP) and GTP (GMP-PNP) analogs.
Number of components: 4

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Component #1: protein, E. coli 50S bound HflX protein in presence of ATP (AMP-P...

ProteinName: E. coli 50S bound HflX protein in presence of ATP (AMP-PNP) and GTP (GMP-PNP) analogs.
Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli) / Strain: BL21

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Component #2: protein, GTPase HflX

ProteinName: GTPase HflX / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 48.392988 kDa
SourceSpecies: Escherichia coli (strain K12) (bacteria) / Strain: K12
Source (engineered)Expression System: Escherichia coli BL21 (bacteria)

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Component #3: nucleic-acid, 5S rRNA

Nucleic-acidName: 5S rRNA5S ribosomal RNA / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
UGCCUGGCGG CCGUAGCGCG GUGGUCCCAC CUGACCCCAU GCCGAACUCA GAAGUGAAAC GCCGUAGCGC CGAUGGUAGU GUGGGGUCUC CCCAUGCGAG AGUAGGGAAC UGCCAGGCAU
MassTheoretical: 38.79009 kDa
SourceSpecies: Escherichia coli (E. coli) / Strain: MRE 600

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Component #4: nucleic-acid, 23S rRNA

Nucleic-acidName: 23S rRNA23S ribosomal RNA / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
GGUUAAGCGA CUAAGCGUAC ACGGUGGAUG CCCUGGCAGU CAGAGGCGAU GAAGGACGUG CUAAUCUGCG AUAAGCGUCG GUAAGGUGAU AUGAACCGUU AUAACCGGCG AUUUCCGAAU GGGGAAACCC AGUGUGUUUC GACACACUAU CAUUAACUGA AUCCAUAGGU UAAUGAGGCG AACCGGGGGA ACUGAAACAU CUAAGUACCC CGAGGAAAAG AAAUCAACCG AGAUUCCCCC AGUAGCGGCG AGCGAACGGG GAGCAGCCCA GAGCCUGAAU CAGUGUGUGU GUUAGUGGAA GCGUCUGGAA AGGCGCGCGA UACAGGGUGA CAGCCCCGUA CACAAAAAUG CACAUGCUGU GAGCUCGAUG AGUAGGGCGG GACACGUGGU AUCCUGUCUG AAUAUGGGGG GACCAUCCUC CAAGGCUAAA UACUCCUGAC UGACCGAUAG UGAACCAGUA CCGUGAGGGA AAGGCGAAAA GAACCCCGGC GAGGGGAGUG AAAAAGAACC UGAAACCGUG UACGUACAAG CAGUGGGAGC ACGCUUAGGC GUGUGACUGC GUACCUUUUG UAUAAUGGGU CAGCGACUUA UAUUCUGUAG CAAGGUUAAC CGAAUAGGGG AGCCGAAGGG AAACCGAGUC UUAACUGGGC GUUAAGUUGC AGGGUAUAGA CCCGAAACCC GGUGAUCUAG CCAUGGGCAG GUUGAAGGUU GGGUAACACU AACUGGAGGA CCGAACCGAC UAAUGUUGAA AAAUUAGCGG AUGACUUGUG GCUGGGGGUG AAAGGCCAAU CAAACCGGGA GAUAGCUGGU UCUCCCCGAA AGCUAUUUAG GUAGCGCCUC GUGAAUUCAU CUCCGGGGGU AGAGCACUGU UUCGGCAAGG GGGUCAUCCC GACUUACCAA CCCGAUGCAA ACUGCGAAUA CCGGAGAAUG UUAUCACGGG AGACACACGG CGGGUGCUAA CGUCCGUCGU GAAGAGGGAA ACAACCCAGA CCGCCAGCUA AGGUCCCAAA GUCAUGGUUA AGUGGGAAAC GAUGUGGGAA GGCCCAGACA GCCAGGAUGU UGGCUUAGAA GCAGCCAUCA UUUAAAGAAA GCGUAAUAGC UCACUGGUCG AGUCGGCCUG CGCGGAAGAU GUAACGGGGC UAAACCAUGC ACCGAAGCUG CGGCAGCGAC GCUUAUGCGU UGUUGGGUAG GGGAGCGUUC UGUAAGCCUG CGAAGGUGUG CUGUGAGGCA UGCUGGAGGU AUCAGAAGUG CGAAUGCUGA CAUAAGUAAC GAUAAAGCGG GUGAAAAGCC CGCUCGCCGG AAGACCAAGG GUUCCUGUCC AACGUUAAUC GGGGCAGGGU GAGUCGACCC CUAAGGCGAG GCCGAAAGGC GUAGUCGAUG GGAAACAGGU UAAUAUUCCU GUACUUGGUG UUACUGCGAA GGGGGGACGG AGAAGGCUAU GUUGGCCGGG CGACGGUUGU CCCGGUUUAA GCGUGUAGGC UGGUUUUCCA GGCAAAUCCG GAAAAUCAAG GCUGAGGCGU GAUGACGAGG CACUACGGUG CUGAAGCAAC AAAUGCCCUG CUUCCAGGAA AAGCCUCUAA GCAUCAGGUA ACAUCAAAUC GUACCCCAAA CCGACACAGG UGGUCAGGUA GAGAAUACCA AGGCGCUUGA GAGAACUCGG GUGAAGGAAC UAGGCAAAAU GGUGCCGUAA CUUCGGGAGA AGGCACGCUG AUAUGUAGGU GAGGUCCCUC GCGGAUGGAG CUGAAAUCAG UCGAAGAUAC CAGCUGGCUG CAACUGUUUA UUAAAAACAC AGCACUGUGC AAACACGAAA GUGGACGUAU ACGGUGUGAC GCCUGCCCGG UGCCGGAAGG UUAAUUGAUG GGGUUAGCGC AAGCGAAGCU CUUGAUCGAA GCCCCGGUAA ACGGCGGCCG UAACUAUAAC GGUCCUAAGG UAGCGAAAUU CCUUGUCGGG UAAGUUCCGA CCUGCACGAA UGGCGUAAUG AUGGCCAGGC UGUCUCCACC CGAGACUCAG UGAAAUUGAA CUCGCUGUGA AGAUGCAGUG UACCCGCGGC AAGACGGAAA GACCCCGUGA ACCUUUACUA UAGCUUGACA CUGAACAUUG AGCCUUGAUG UGUAGGAUAG GUGGGAGGCU UUGAAGUGUG GACGCCAGUC UGCAUGGAGC CGACCUUGAA AUACCACCCU UUAAUGUUUG AUGUUCUAAC GUUGACCCGU AAUCCGGGUU GCGGACAGUG UCUGGUGGGU AGUUUGACUG GGGCGGUCUC CUCCUAAAGA GUAACGGAGG AGCACGAAGG UUGGCUAAUC CUGGUCGGAC AUCAGGAGGU UAGUGCAAUG GCAUAAGCCA GCUUGACUGC GAGCGUGACG GCGCGAGCAG GUGCGAAAGC AGGUCAUAGU GAUCCGGUGG UUCUGAAUGG AAGGGCCAUC GCUCAACGGA UAAAAGGUAC UCCGGGGAUA ACAGGCUGAU ACCGCCCAAG AGUUCAUAUC GACGGCGGUG UUUGGCACCU CGAUGUCGGC UCAUCACAUC CUGGGGCUGA AGUAGGUCCC AAGGGUAUGG CUGUUCGCCA UUUAAAGUGG UACGCGAGCU GGGUUUAGAA CGUCGUGAGA CAGUUCGGUC CCUAUCUGCC GUGGGCGCUG GAGAACUGAG GGGGGCUGCU CCUAGUACGA GAGGACCGGA GUGGACGCAU CACUGGUGUU CGGGUUGUCA UGCCAAUGGC ACUGCCCGGU AGCUAAAUGC GGAAGAGAUA AGUGCUGAAA GCAUCUAAGC ACGAAACUUG CCCCGAGAUG AGUUCUCCCU GACCCUUUAA GGGUCCUGAA GGAACGUUGA AGACGACGAC GUUGAUAGGC CGGGUGUGUA AGCGCAGCGA UGCGUUGAGC UAACCGGUAC UAAUGAACCG UGAGGCUUAA CCU
MassTheoretical: 941.306188 kDa
SourceSpecies: Escherichia coli (E. coli) / Strain: MRE 600

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionpH: 7.5
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 278 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
ImagingMicroscope: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 2 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: DIFFRACTION
Specimen HolderModel: GATAN LIQUID NITROGEN
CameraDetector: FEI EAGLE (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 61557
3D reconstructionResolution: 8.1 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Output model

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