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- EMDB-15931: Cryo-EM structure of MLE in complex with ADP:AlF4 and U10 RNA -

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Basic information

Entry
Database: EMDB / ID: EMD-15931
TitleCryo-EM structure of MLE in complex with ADP:AlF4 and U10 RNA
Map data
Sample
  • Complex: MLE in complex with ADP:AlF4 and U10 RNA
    • Protein or peptide: Dosage compensation regulator
    • RNA: RNA (5'-R(P*UP*UP*UP*UP*UP*UP*UP*UP*UP*UP*U)-3')
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: TETRAFLUOROALUMINATE ION
  • Ligand: water
KeywordsRNA Helicase / Drosophila dosage compensation / RNA BINDING PROTEIN
Function / homology
Function and homology information


RIP-mediated NFkB activation via ZBP1 / X chromosome located dosage compensation complex, transcription activating / 3'-5' DNA/RNA helicase activity / DEx/H-box helicases activate type I IFN and inflammatory cytokines production / male courtship behavior, veined wing generated song production / regulation of cytoplasmic translation / regulatory region RNA binding / sex-chromosome dosage compensation / PKR-mediated signaling / MSL complex ...RIP-mediated NFkB activation via ZBP1 / X chromosome located dosage compensation complex, transcription activating / 3'-5' DNA/RNA helicase activity / DEx/H-box helicases activate type I IFN and inflammatory cytokines production / male courtship behavior, veined wing generated song production / regulation of cytoplasmic translation / regulatory region RNA binding / sex-chromosome dosage compensation / PKR-mediated signaling / MSL complex / dosage compensation by hyperactivation of X chromosome / positive regulation of DNA topoisomerase (ATP-hydrolyzing) activity / polytene chromosome / 3'-5' RNA helicase activity / regulation of mRNA processing / axon extension / lncRNA binding / 3'-5' DNA helicase activity / DNA duplex unwinding / nuclear chromosome / positive regulation of heterochromatin formation / X chromosome / DNA helicase activity / helicase activity / determination of adult lifespan / double-stranded RNA binding / chromosome / double-stranded DNA binding / RNA helicase activity / RNA helicase / ribonucleoprotein complex / chromatin binding / chromatin / nucleolus / ATP hydrolysis activity / positive regulation of transcription by RNA polymerase II / RNA binding / ATP binding / nucleus / cytosol
Similarity search - Function
DHX9, first double-stranded RNA binding domain / DHX9, second double-stranded RNA binding domain / DHX9, DEXH-box helicase domain / : / Helicase associated domain (HA2), ratchet-like / DEAD-box helicase, OB fold / Oligonucleotide/oligosaccharide-binding (OB)-fold / Helicase-associated domain / Helicase associated domain (HA2), winged-helix / Helicase associated domain (HA2) Add an annotation ...DHX9, first double-stranded RNA binding domain / DHX9, second double-stranded RNA binding domain / DHX9, DEXH-box helicase domain / : / Helicase associated domain (HA2), ratchet-like / DEAD-box helicase, OB fold / Oligonucleotide/oligosaccharide-binding (OB)-fold / Helicase-associated domain / Helicase associated domain (HA2), winged-helix / Helicase associated domain (HA2) Add an annotation / Double-stranded RNA binding motif / Double-stranded RNA binding motif / DNA/RNA helicase, ATP-dependent, DEAH-box type, conserved site / DEAH-box subfamily ATP-dependent helicases signature. / Double stranded RNA-binding domain (dsRBD) profile. / Double-stranded RNA-binding domain / DEAD/DEAH box helicase / DEAD/DEAH box helicase domain / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Dosage compensation regulator
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.86 Å
AuthorsJagtap PKA / Hennig J
Funding support Germany, 1 items
OrganizationGrant numberCountry
EIPOD fellowship under Marie Sklodowska-Curie Actions COFUND Germany
CitationJournal: Mol Cell / Year: 2023
Title: Structural basis of RNA-induced autoregulation of the DExH-type RNA helicase maleless.
Authors: Pravin Kumar Ankush Jagtap / Marisa Müller / Anna E Kiss / Andreas W Thomae / Karine Lapouge / Martin Beck / Peter B Becker / Janosch Hennig /
Abstract: RNA unwinding by DExH-type helicases underlies most RNA metabolism and function. It remains unresolved if and how the basic unwinding reaction of helicases is regulated by auxiliary domains. We ...RNA unwinding by DExH-type helicases underlies most RNA metabolism and function. It remains unresolved if and how the basic unwinding reaction of helicases is regulated by auxiliary domains. We explored the interplay between the RecA and auxiliary domains of the RNA helicase maleless (MLE) from Drosophila using structural and functional studies. We discovered that MLE exists in a dsRNA-bound open conformation and that the auxiliary dsRBD2 domain aligns the substrate RNA with the accessible helicase tunnel. In an ATP-dependent manner, dsRBD2 associates with the helicase module, leading to tunnel closure around ssRNA. Furthermore, our structures provide a rationale for blunt-ended dsRNA unwinding and 3'-5' translocation by MLE. Structure-based MLE mutations confirm the functional relevance of our model for RNA unwinding. Our findings contribute to our understanding of the fundamental mechanics of auxiliary domains in DExH helicase MLE, which serves as a model for its human ortholog and potential therapeutic target, DHX9/RHA.
History
DepositionOct 6, 2022-
Header (metadata) releaseOct 18, 2023-
Map releaseOct 18, 2023-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15931.png

Downloads & links

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Map

FileDownload / File: emd_15931.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.822 Å
Density
Contour LevelBy AUTHOR: 0.0951
Minimum - Maximum-0.15522125 - 0.5735441
Average (Standard dev.)0.0051008356 (±0.018691814)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 210.432 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15931_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_15931_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_15931_half_map_2.map
Projections & Slices
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Slices (1/2)
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Histogram (log scale)

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Sample components

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Entire : MLE in complex with ADP:AlF4 and U10 RNA

EntireName: MLE in complex with ADP:AlF4 and U10 RNA
Components
  • Complex: MLE in complex with ADP:AlF4 and U10 RNA
    • Protein or peptide: Dosage compensation regulator
    • RNA: RNA (5'-R(P*UP*UP*UP*UP*UP*UP*UP*UP*UP*UP*U)-3')
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: TETRAFLUOROALUMINATE ION
  • Ligand: water

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Supramolecule #1: MLE in complex with ADP:AlF4 and U10 RNA

SupramoleculeName: MLE in complex with ADP:AlF4 and U10 RNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 133.2 KDa

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Macromolecule #1: Dosage compensation regulator

MacromoleculeName: Dosage compensation regulator / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA helicase
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 130.46507 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDIKSFLYQF CAKSQIEPKF DIRQTGPKNR QRFLCEVRVE PNTYIGVGNS TNKKDAEKNA CRDFVNYLVR VGKLNTNDVP ADAGASGGG PRTGLEGAGM AGGSGQQKRV FDGQSGPQDL GEAYRPLNHD GGDGGNRYSV IDRIQEQRDM NEAEAFDVNA A IHGNWTIE ...String:
MDIKSFLYQF CAKSQIEPKF DIRQTGPKNR QRFLCEVRVE PNTYIGVGNS TNKKDAEKNA CRDFVNYLVR VGKLNTNDVP ADAGASGGG PRTGLEGAGM AGGSGQQKRV FDGQSGPQDL GEAYRPLNHD GGDGGNRYSV IDRIQEQRDM NEAEAFDVNA A IHGNWTIE NAKERLNIYK QTNNIRDDYK YTPVGPEHAR SFLAELSIYV PALNRTVTAR ESGSNKKSAS KSCALSLVRQ LF HLNVIEP FSGTLKKKKD EQLKPYPVKL SPNLINKIDE VIKGLDLPVV NPRNIKIELD GPPIPLIVNL SRIDSSQQDG EKR QESSVI PWAPPQANWN TWHACNIDEG ELATTSIDDL SMDYERSLRD RRQNDNEYRQ FLEFREKLPI AAMRSEILTA INDN PVVII RGNTGCGKTT QIAQYILDDY ICSGQGGYAN IYVTQPRRIS AISVAERVAR ERCEQLGDTV GYSVRFESVF PRPYG AILF CTVGVLLRKL EAGLRGVSHI IVDEIHERDV NSDFLLVILR DMVDTYPDLH VILMSATIDT TKFSKYFGIC PVLEVP GRA FPVQQFFLED IIQMTDFVPS AESRRKRKEV EDEEQLLSED KDEAEINYNK VCEDKYSQKT RNAMAMLSES DVSFELL EA LLMHIKSKNI PGAILVFLPG WNLIFALMKF LQNTNIFGDT SQYQILPCHS QIPRDEQRKV FEPVPEGVTK IILSTNIA E TSITIDDIVF VIDICKARMK LFTSHNNLTS YATVWASKTN LEQRKGRAGR VRPGFCFTLC SRARFQALED NLTPEMFRT PLHEMALTIK LLRLGSIHHF LSKALEPPPV DAVIEAEVLL REMRCLDAND ELTPLGRLLA RLPIEPRLGK MMVLGAVFGC ADLMAIMAS YSSTFSEVFS LDIGQRRLAN HQKALSGTKC SDHVAMIVAS QMWRREKQRG EHMEARFCDW KGLQMSTMNV I WDAKQQLL DLLQQAGFPE ECMISHEVDE RIDGDDPVLD VSLALLCLGL YPNICVHKEK RKVLTTESKA ALLHKTSVNC SN LAVTFPY PFFVFGEKIR TRAVSCKQLS MVSPLQVILF GSRKIDLAAN NIVRVDNWLN FDIEPELAAK IGALKPALED LIT VACDNP SDILRLEEPY AQLVKVVKDL CVKSAGDFGL QRE

UniProtKB: Dosage compensation regulator

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Macromolecule #2: RNA (5'-R(P*UP*UP*UP*UP*UP*UP*UP*UP*UP*UP*U)-3')

MacromoleculeName: RNA (5'-R(P*UP*UP*UP*UP*UP*UP*UP*UP*UP*UP*U)-3') / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 3.0167 KDa
SequenceString:
UUUUUUUUUU

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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Macromolecule #4: TETRAFLUOROALUMINATE ION

MacromoleculeName: TETRAFLUOROALUMINATE ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: ALF
Molecular weightTheoretical: 102.975 Da
Chemical component information

ChemComp-ALF:
TETRAFLUOROALUMINATE ION

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 5 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

Concentration0.612 mg/mL
BufferpH: 7.5
Details: 20 mM Tris pH 7.5, 50 mM NaCl, 1mM DTT, 1mM ADP, 1mM AlF3, 10 mM NaF, 2mM MgCl2, 0.5% glycerol, 0.005% Triton X-100
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 279.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 61.2 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 559766
FSC plot (resolution estimation)

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