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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: EMD-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.
Sample
  • Complex: E. coli 50S bound HflX protein in presence of ATP (AMP-PNP) and GTP (GMP-PNP) analogs.
    • Protein or peptide: GTPase HflX
    • RNA: 5S rRNA5S ribosomal RNA
    • RNA: 23S rRNA23S ribosomal RNA
KeywordsATPase / RNA helicase / Heat stress / RIBOSOME
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 / 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 / GTP binding domain / EF-G domain III/V-like / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Biological speciesEscherichia coli (E. coli) / Escherichia coli (strain K12) (bacteria)
Methodsingle 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, 2018-
Header (metadata) releaseJun 27, 2018-
Map releaseJun 27, 2018-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2
  • Imaged by UCSF Chimera
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  • 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

emd_6979.png

Downloads & links

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Map

FileDownload / File: emd_6979.map.gz / Format: CCP4 / Size: 35.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationE. coli 50S bound HflX protein in presence of ATP (AMP-PNP) and GTP (GMP-PNP) analogs.
Voxel sizeX=Y=Z: 1.89 Å
Density
Contour LevelBy AUTHOR: 2.0 / Movie #1: 2
Minimum - Maximum-7.187189 - 15.866949
Average (Standard dev.)0.081063196 (±0.78008014)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions210210210
Spacing210210210
CellA=B=C: 396.9 Å
α=β=γ: 90.0 °

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
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.
Components
  • Complex: E. coli 50S bound HflX protein in presence of ATP (AMP-PNP) and GTP (GMP-PNP) analogs.
    • Protein or peptide: GTPase HflX
    • RNA: 5S rRNA5S ribosomal RNA
    • RNA: 23S rRNA23S ribosomal RNA

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Supramolecule #1: E. coli 50S bound HflX protein in presence of ATP (AMP-PNP) and G...

SupramoleculeName: E. coli 50S bound HflX protein in presence of ATP (AMP-PNP) and GTP (GMP-PNP) analogs.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli) / Strain: BL21

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Macromolecule #1: GTPase HflX

MacromoleculeName: GTPase HflX / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 48.392988 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MFDRYDAGEQ AVLVHIYFTQ DKDMEDLQEF ESLVSSAGVE ALQVITGSRK APHPKYFVGE GKAVEIAEAV KATGASVVLF DHALSPAQE RNLERLCECR VIDRTGLILD IFAQRARTHE GKLQVELAQL RHLATRLVRG WTHLERQKGG IGLRGPGETQ L ETDRRLLR ...String:
MFDRYDAGEQ AVLVHIYFTQ DKDMEDLQEF ESLVSSAGVE ALQVITGSRK APHPKYFVGE GKAVEIAEAV KATGASVVLF DHALSPAQE RNLERLCECR VIDRTGLILD IFAQRARTHE GKLQVELAQL RHLATRLVRG WTHLERQKGG IGLRGPGETQ L ETDRRLLR NRIVQIQSRL ERVEKQREQG RQSRIKADVP TVSLVGYTNA GKSTLFNRIT EARVYAADQL FATLDPTLRR ID VADVGET VLADTVGFIR HLPHDLVAAF KATLQETRQA TLLLHVIDAA DVRVQENIEA VNTVLEEIDA HEIPTLLVMN KID MLEDFE PRIDRDEENK PNRVWLSAQT GAGIPQLFQA LTERLSGEVA QHTLRLPPQE GRLRSRFYQL QAIEKEWMEE DGSV SLQVR MPIVDWRRLC KQEPALIDYL I

UniProtKB: GTPase HflX

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Macromolecule #2: 5S rRNA

MacromoleculeName: 5S rRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MRE 600
Molecular weightTheoretical: 38.79009 KDa
SequenceString:
UGCCUGGCGG CCGUAGCGCG GUGGUCCCAC CUGACCCCAU GCCGAACUCA GAAGUGAAAC GCCGUAGCGC CGAUGGUAGU GUGGGGUCU CCCCAUGCGA GAGUAGGGAA CUGCCAGGCA U

GENBANK: GENBANK: CP027586.1

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Macromolecule #3: 23S rRNA

MacromoleculeName: 23S rRNA / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MRE 600
Molecular weightTheoretical: 941.306188 KDa
SequenceString: GGUUAAGCGA CUAAGCGUAC ACGGUGGAUG CCCUGGCAGU CAGAGGCGAU GAAGGACGUG CUAAUCUGCG AUAAGCGUCG GUAAGGUGA UAUGAACCGU UAUAACCGGC GAUUUCCGAA UGGGGAAACC CAGUGUGUUU CGACACACUA UCAUUAACUG A AUCCAUAG ...String:
GGUUAAGCGA CUAAGCGUAC ACGGUGGAUG CCCUGGCAGU CAGAGGCGAU GAAGGACGUG CUAAUCUGCG AUAAGCGUCG GUAAGGUGA UAUGAACCGU UAUAACCGGC GAUUUCCGAA UGGGGAAACC CAGUGUGUUU CGACACACUA UCAUUAACUG A AUCCAUAG GUUAAUGAGG CGAACCGGGG GAACUGAAAC AUCUAAGUAC CCCGAGGAAA AGAAAUCAAC CGAGAUUCCC CC AGUAGCG GCGAGCGAAC GGGGAGCAGC CCAGAGCCUG AAUCAGUGUG UGUGUUAGUG GAAGCGUCUG GAAAGGCGCG CGA UACAGG GUGACAGCCC CGUACACAAA AAUGCACAUG CUGUGAGCUC GAUGAGUAGG GCGGGACACG UGGUAUCCUG UCUG AAUAU GGGGGGACCA UCCUCCAAGG CUAAAUACUC CUGACUGACC GAUAGUGAAC CAGUACCGUG AGGGAAAGGC GAAAA GAAC CCCGGCGAGG GGAGUGAAAA AGAACCUGAA ACCGUGUACG UACAAGCAGU GGGAGCACGC UUAGGCGUGU GACUGC GUA CCUUUUGUAU AAUGGGUCAG CGACUUAUAU UCUGUAGCAA GGUUAACCGA AUAGGGGAGC CGAAGGGAAA CCGAGUC UU AACUGGGCGU UAAGUUGCAG GGUAUAGACC CGAAACCCGG UGAUCUAGCC AUGGGCAGGU UGAAGGUUGG GUAACACU A ACUGGAGGAC CGAACCGACU AAUGUUGAAA AAUUAGCGGA UGACUUGUGG CUGGGGGUGA AAGGCCAAUC AAACCGGGA GAUAGCUGGU UCUCCCCGAA AGCUAUUUAG GUAGCGCCUC GUGAAUUCAU CUCCGGGGGU AGAGCACUGU UUCGGCAAGG GGGUCAUCC CGACUUACCA ACCCGAUGCA AACUGCGAAU ACCGGAGAAU GUUAUCACGG GAGACACACG GCGGGUGCUA A CGUCCGUC GUGAAGAGGG AAACAACCCA GACCGCCAGC UAAGGUCCCA AAGUCAUGGU UAAGUGGGAA ACGAUGUGGG AA GGCCCAG ACAGCCAGGA UGUUGGCUUA GAAGCAGCCA UCAUUUAAAG AAAGCGUAAU AGCUCACUGG UCGAGUCGGC CUG CGCGGA AGAUGUAACG GGGCUAAACC AUGCACCGAA GCUGCGGCAG CGACGCUUAU GCGUUGUUGG GUAGGGGAGC GUUC UGUAA GCCUGCGAAG GUGUGCUGUG AGGCAUGCUG GAGGUAUCAG AAGUGCGAAU GCUGACAUAA GUAACGAUAA AGCGG GUGA AAAGCCCGCU CGCCGGAAGA CCAAGGGUUC CUGUCCAACG UUAAUCGGGG CAGGGUGAGU CGACCCCUAA GGCGAG GCC GAAAGGCGUA GUCGAUGGGA AACAGGUUAA UAUUCCUGUA CUUGGUGUUA CUGCGAAGGG GGGACGGAGA AGGCUAU GU UGGCCGGGCG ACGGUUGUCC CGGUUUAAGC GUGUAGGCUG GUUUUCCAGG CAAAUCCGGA AAAUCAAGGC UGAGGCGU G AUGACGAGGC ACUACGGUGC UGAAGCAACA AAUGCCCUGC UUCCAGGAAA AGCCUCUAAG CAUCAGGUAA CAUCAAAUC GUACCCCAAA CCGACACAGG UGGUCAGGUA GAGAAUACCA AGGCGCUUGA GAGAACUCGG GUGAAGGAAC UAGGCAAAAU GGUGCCGUA ACUUCGGGAG AAGGCACGCU GAUAUGUAGG UGAGGUCCCU CGCGGAUGGA GCUGAAAUCA GUCGAAGAUA C CAGCUGGC UGCAACUGUU UAUUAAAAAC ACAGCACUGU GCAAACACGA AAGUGGACGU AUACGGUGUG ACGCCUGCCC GG UGCCGGA AGGUUAAUUG AUGGGGUUAG CGCAAGCGAA GCUCUUGAUC GAAGCCCCGG UAAACGGCGG CCGUAACUAU AAC GGUCCU AAGGUAGCGA AAUUCCUUGU CGGGUAAGUU CCGACCUGCA CGAAUGGCGU AAUGAUGGCC AGGCUGUCUC CACC CGAGA CUCAGUGAAA UUGAACUCGC UGUGAAGAUG CAGUGUACCC GCGGCAAGAC GGAAAGACCC CGUGAACCUU UACUA UAGC UUGACACUGA ACAUUGAGCC UUGAUGUGUA GGAUAGGUGG GAGGCUUUGA AGUGUGGACG CCAGUCUGCA UGGAGC CGA CCUUGAAAUA CCACCCUUUA AUGUUUGAUG UUCUAACGUU GACCCGUAAU CCGGGUUGCG GACAGUGUCU GGUGGGU AG UUUGACUGGG GCGGUCUCCU CCUAAAGAGU AACGGAGGAG CACGAAGGUU GGCUAAUCCU GGUCGGACAU CAGGAGGU U AGUGCAAUGG CAUAAGCCAG CUUGACUGCG AGCGUGACGG CGCGAGCAGG UGCGAAAGCA GGUCAUAGUG AUCCGGUGG UUCUGAAUGG AAGGGCCAUC GCUCAACGGA UAAAAGGUAC UCCGGGGAUA ACAGGCUGAU ACCGCCCAAG AGUUCAUAUC GACGGCGGU GUUUGGCACC UCGAUGUCGG CUCAUCACAU CCUGGGGCUG AAGUAGGUCC CAAGGGUAUG GCUGUUCGCC A UUUAAAGU GGUACGCGAG CUGGGUUUAG AACGUCGUGA GACAGUUCGG UCCCUAUCUG CCGUGGGCGC UGGAGAACUG AG GGGGGCU GCUCCUAGUA CGAGAGGACC GGAGUGGACG CAUCACUGGU GUUCGGGUUG UCAUGCCAAU GGCACUGCCC GGU AGCUAA AUGCGGAAGA GAUAAGUGCU GAAAGCAUCU AAGCACGAAA CUUGCCCCGA GAUGAGUUCU CCCUGACCCU UUAA GGGUC CUGAAGGAAC GUUGAAGACG ACGACGUUGA UAGGCCGGGU GUGUAAGCGC AGCGAUGCGU UGAGCUAACC GGUAC UAAU GAACCGUGAG GCUUAACCU

GENBANK: GENBANK: CP016018.1

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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
GridMaterial: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION
Sample stageSpecimen holder model: GATAN 910 MULTI-SPECIMEN SINGLE TILT CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI EAGLE (4k x 4k) / Number grids imaged: 4 / Average exposure time: 2.0 sec. / Average electron dose: 20.0 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

1395


Details: The only 50S density was isolated from 70S density and filtered to very low resolution using SPIDER.
Initial angle assignmentType: PROJECTION MATCHING
Projection matching processing - Number reference projections: 83
Software - Name: SPIDER (ver. 23.03)
Final angle assignmentType: PROJECTION MATCHING
Projection matching processing - Number reference projections: 83
Final reconstructionResolution.type: BY AUTHOR / Resolution: 8.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: EMAN2 (ver. 2.2 final) / Number images used: 61557
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT
Output model

PDB-5zzm:
E. coli 50S subunit bound HflX protein in presence of ATP (AMP-PNP) and GTP (GMP-PNP) analogs.

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