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- EMDB-11788: Cryo-EM structure of the RUVBL1-RUVBL2-DHX34 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-11788
TitleCryo-EM structure of the RUVBL1-RUVBL2-DHX34 complex
Map dataCryo-EM reconstruction of RUVBL1-RUVBL2 bound to DHX34 helicase
Sample
  • Complex: RUVBL1-RUVBL2-DHX34 complex
    • Protein or peptide: RUVBL1
    • Protein or peptide: RUVBL2
    • Protein or peptide: DHX34
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.97 Å
AuthorsLopez-Perrote A / Rodriguez CF / Llorca O
Funding support Spain, 3 items
OrganizationGrant numberCountry
Spanish Ministry of Science, Innovation, and UniversitiesSAF2017-82632-P Spain
Other governmentP2018/NMT4443 Spain
Other governmentY2018/BIO4747 Spain
CitationJournal: Elife / Year: 2020
Title: Regulation of RUVBL1-RUVBL2 AAA-ATPases by the nonsense-mediated mRNA decay factor DHX34, as evidenced by Cryo-EM.
Authors: Andres López-Perrote / Nele Hug / Ana González-Corpas / Carlos F Rodríguez / Marina Serna / Carmen García-Martín / Jasminka Boskovic / Rafael Fernandez-Leiro / Javier F Caceres / Oscar Llorca /
Abstract: Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that degrades aberrant mRNAs and also regulates the expression of a wide range of physiological transcripts. RUVBL1 and RUVBL2 AAA-ATPases ...Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that degrades aberrant mRNAs and also regulates the expression of a wide range of physiological transcripts. RUVBL1 and RUVBL2 AAA-ATPases form an hetero-hexameric ring that is part of several macromolecular complexes such as INO80, SWR1, and R2TP. Interestingly, RUVBL1-RUVBL2 ATPase activity is required for NMD activation by an unknown mechanism. Here, we show that DHX34, an RNA helicase regulating NMD initiation, directly interacts with RUVBL1-RUVBL2 in vitro and in cells. Cryo-EM reveals that DHX34 induces extensive changes in the N-termini of every RUVBL2 subunit in the complex, stabilizing a conformation that does not bind nucleotide and thereby down-regulates ATP hydrolysis of the complex. Using ATPase-deficient mutants, we find that DHX34 acts exclusively on the RUVBL2 subunits. We propose a model, where DHX34 acts to couple RUVBL1-RUVBL2 ATPase activity to the assembly of factors required to initiate the NMD response.
History
DepositionSep 25, 2020-
Header (metadata) releaseNov 25, 2020-
Map releaseNov 25, 2020-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.012
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11788.png

Downloads & links

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Map

FileDownload / File: emd_11788.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM reconstruction of RUVBL1-RUVBL2 bound to DHX34 helicase
Voxel sizeX=Y=Z: 1.047 Å
Density
Contour LevelBy AUTHOR: 0.012 / Movie #1: 0.012
Minimum - Maximum-0.017267019 - 0.26354554
Average (Standard dev.)0.00043892732 (±0.0045909053)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 293.16 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0471.0471.047
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z293.160293.160293.160
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS280280280
D min/max/mean-0.0170.2640.000

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Supplemental data

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Sample components

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Entire : RUVBL1-RUVBL2-DHX34 complex

EntireName: RUVBL1-RUVBL2-DHX34 complex
Components
  • Complex: RUVBL1-RUVBL2-DHX34 complex
    • Protein or peptide: RUVBL1
    • Protein or peptide: RUVBL2
    • Protein or peptide: DHX34

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Supramolecule #1: RUVBL1-RUVBL2-DHX34 complex

SupramoleculeName: RUVBL1-RUVBL2-DHX34 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Complex of human RUVBL1-RUVBL2 hexamer bound to DHX34 helicase
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 (DE3)
Molecular weightTheoretical: 0.44928 kDa/nm

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Macromolecule #1: RUVBL1

MacromoleculeName: RUVBL1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MADLNWISAG HAIADVGTMA TVTATTKVPE IRDVTRIERI GAHSHIRGLG LDDALEPRQA SQGMVGQLAA RRAAGVVLEM IREGKIAGRA VLIAGQPGTG KTAIAMGMAQ ALGPDTPFTA IAGSEIFSLE MSKTEALTQA FRRSIGVRIK EETEIIEGEV VEIQIDRPAT ...String:
MADLNWISAG HAIADVGTMA TVTATTKVPE IRDVTRIERI GAHSHIRGLG LDDALEPRQA SQGMVGQLAA RRAAGVVLEM IREGKIAGRA VLIAGQPGTG KTAIAMGMAQ ALGPDTPFTA IAGSEIFSLE MSKTEALTQA FRRSIGVRIK EETEIIEGEV VEIQIDRPAT GTGSKVGKLT LKTTEMETIY DLGTKMIESL TKDKVQAGDV ITIDKATGKI SKLGRSFTRA RDYDAMGSQT KFVQCPDGEL QKRKEVVHTV SLHEIDVINS RTQGFLALFS GDTGEIKSEV REQINAKVAE WREEGKAEII PGVLFIDEVH MLDIESFSFL NRALESDMAP VLIMATNRGI TRIRGTSYQS PHGIPIDLLD RLLIVSTTPY SEKDTKQILR IRCEEEDVEM SEDAYTVLTR IGLETSLRYA IQLITAASLV CRKRKGTEVQ VDDIKRVYSL FLDESRSTQY MKEYQDAFLF NELKGETMDT S

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Macromolecule #2: RUVBL2

MacromoleculeName: RUVBL2 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO / EC number: DNA helicase
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MADLNWISAG HAIADVGTMA TVTATTKVPE IRDVTRIERI GAHSHIRGLG LDDALEPRQA SQGMVGQLAA RRAAGVVLEM IREGKIAGRA VLIAGQPGTG KTAIAMGMAQ ALGPDTPFTA IAGSEIFSLE MSKTEALTQA FRRSIGVRIK EETEIIEGEV VEIQIDRPAT ...String:
MADLNWISAG HAIADVGTMA TVTATTKVPE IRDVTRIERI GAHSHIRGLG LDDALEPRQA SQGMVGQLAA RRAAGVVLEM IREGKIAGRA VLIAGQPGTG KTAIAMGMAQ ALGPDTPFTA IAGSEIFSLE MSKTEALTQA FRRSIGVRIK EETEIIEGEV VEIQIDRPAT GTGSKVGKLT LKTTEMETIY DLGTKMIESL TKDKVQAGDV ITIDKATGKI SKLGRSFTRA RDYDAMGSQT KFVQCPDGEL QKRKEVVHTV SLHEIDVINS RTQGFLALFS GDTGEIKSEV REQINAKVAE WREEGKAEII PGVLFIDEVH MLDIESFSFL NRALESDMAP VLIMATNRGI TRIRGTSYQS PHGIPIDLLD RLLIVSTTPY SEKDTKQILR IRCEEEDVEM SEDAYTVLTR IGLETSLRYA IQLITAASLV CRKRKGTEVQ VDDIKRVYSL FLDESRSTQY MKEYQDAFLF NELKGETMDT S

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Macromolecule #3: DHX34

MacromoleculeName: DHX34 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO / EC number: RNA helicase
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Mammalian expression vector Flag-MCS-pcDNA3.1 (others)
SequenceString: MDYKDHDGDY KDHDIDYKDD DDKEFCRYPA QWRPLEPPPR TREGRDRRDH HRAPSEEEAL EKWDWNCPE TRRLLEDAFF REEDYIRQGS EECQKFWTFF ERLQRFQNLK TSRKEEKDPG Q PKHSIPAL ADLPRTYDPR YRINLSVLGP ATRGSQGLGR HLPAERVAEF ...String:
MDYKDHDGDY KDHDIDYKDD DDKEFCRYPA QWRPLEPPPR TREGRDRRDH HRAPSEEEAL EKWDWNCPE TRRLLEDAFF REEDYIRQGS EECQKFWTFF ERLQRFQNLK TSRKEEKDPG Q PKHSIPAL ADLPRTYDPR YRINLSVLGP ATRGSQGLGR HLPAERVAEF RRALLHYLDF GQ KQAFGRL AKLQRERAAL PIAQYGNRIL QTLKEHQVVV VAGDTGCGKS TQVPQYLLAA GFS HVACTQ PRRIACISLA KRVGFESLSQ YGSQVGYQIR FESTRSAATK IVFLTVGLLL RQIQ REPSL PQYEVLIVDE VHERHLHNDF LLGVLQRLLP TRPDLKVILM SATINISLFS SYFSN APVV QVPGRLFPIT VVYQPQEAEP TTSKSEKLDP RPFLRVLESI DHKYPPEERG DLLVFL SGM AEISAVLEAA QTYASHTQRW VVLPLHSALS VADQDKVFDV APPGVRKCIL STNIAET SV TIDGIRFVVD SGKVKEMSYD PQAKLQRLQE FWISQASAEQ RKGRAGRTGP GVCFRLYA E SDYDAFAPYP VPEIRRVALD SLVLQMKSMS VGDPRTFPFI EPPPPASLET AILYLRDQG ALDSSEALTP IGSLLAQLPV DVVIGKMLIL GSMFSLVEPV LTIAAALSVQ SPFTRSAQSS PECAAARRP LESDQGDPFT LFNVFNAWVQ VKSERSRNSR KWCRRRGIED HRLYEMANLR R QFKELLED HGLLAGAQAA QVGDSYSRLQ QRRERRALHQ LKRQHEEGAG RRRKVLRLQE EQ DGGSSDE DRAGPAPPGA SDGVDIQDVK FKLRHDLAQL QAAASSAQDL SCEQLALLKL VLG RGLYPQ LAVPDAFNSS RKDSDQIFHT QAKQGAVLHP TCVFAGSPEV LHAQELEASN CDGS RDDKD KMSSKHQLLS FVSLLETNKP YLVNCVRIPA LQSLLLFSRS LDTNGDCSRL VADGW LELQ LADSESAIRL LAASLRLRAR WESALDRQLA HQAQQQLEEE EEDTPVSPKE VATLSK ELL QFTASKIPYS LRRLTGLEVQ NMYVGPQTIP ATPHLPGLFG SSTLSPHPTK GGYAVTD FL TYNCLTNDTD LYSDCLRTFW TCPHCGLHAP LTPLERIAHE NTCPQAPQDG PPGAEEAA L ETLQKTSVLQ RPYHCEACGK DFLFTPTEVL RHRKQHV

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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

Concentration0.35 mg/mL
BufferpH: 7.4 / Component - Formula: Tris-NaCl / Component - Name: TBS / Details: 50 mM Tris-HCl pH 7.4, 150 mM NaCl
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 3047 / Average electron dose: 48.1 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 47756 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 0.003 µm / Nominal defocus min: 0.0015 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 353057
CTF correctionSoftware - Name: RELION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.97 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 41237
Initial angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION
FSC plot (resolution estimation)

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