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- EMDB-62234: Cryo-EM structure of DCL4-Hairpin RNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-62234
TitleCryo-EM structure of DCL4-Hairpin RNA complex
Map data
Sample
  • Complex: DCL4-RNA
    • Protein or peptide: Dicer-like protein 4
    • RNA: RNA (70-MER)
  • Ligand: MAGNESIUM ION
KeywordsDCL4 / DRB4 / siRNA / Dicer / RNA BINDING PROTEIN/RNA / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


vegetative phase change / RNAi-mediated antiviral immunity against RNA virus / lsiRNA processing / ta-siRNA processing / RNAi-mediated antiviral immune response / Hydrolases; Acting on ester bonds; Endoribonucleases producing 5'-phosphomonoesters / ribonuclease III activity / siRNA processing / negative regulation of gene expression via chromosomal CpG island methylation / helicase activity ...vegetative phase change / RNAi-mediated antiviral immunity against RNA virus / lsiRNA processing / ta-siRNA processing / RNAi-mediated antiviral immune response / Hydrolases; Acting on ester bonds; Endoribonucleases producing 5'-phosphomonoesters / ribonuclease III activity / siRNA processing / negative regulation of gene expression via chromosomal CpG island methylation / helicase activity / DNA-templated transcription termination / cytoplasmic ribonucleoprotein granule / defense response to virus / RNA binding / ATP binding / metal ion binding / nucleus / cytoplasm / cytosol
Similarity search - Function
double strand RNA binding domain from DEAD END PROTEIN 1 / Dicer dimerisation domain / Dicer dimerisation domain superfamily / Dicer dimerisation domain / Dicer double-stranded RNA-binding fold domain profile. / Ribonuclease III family signature. / Ribonuclease III domain / Ribonuclease III family domain profile. / Ribonuclease III family / Ribonuclease III domain ...double strand RNA binding domain from DEAD END PROTEIN 1 / Dicer dimerisation domain / Dicer dimerisation domain superfamily / Dicer dimerisation domain / Dicer double-stranded RNA-binding fold domain profile. / Ribonuclease III family signature. / Ribonuclease III domain / Ribonuclease III family domain profile. / Ribonuclease III family / Ribonuclease III domain / PAZ / PAZ domain profile. / PAZ domain / Double-stranded RNA binding motif / Ribonuclease III, endonuclease domain superfamily / Double stranded RNA-binding domain (dsRBD) profile. / Double-stranded RNA-binding domain / DEAD/DEAH box helicase domain / DEAD/DEAH box helicase / 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
Dicer-like protein 4
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.15 Å
AuthorsWang C / Du J
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32325008 China
CitationJournal: Nat Plants / Year: 2026
Title: Molecular basis of DRB4-assisted long RNA processing and 21-nucleotide siRNA biogenesis by DCL4 in plants.
Authors: Changshi Wang / Cheng Chi / Yuelin Liu / Jun Zhao / Qian Wang / Nana Wang / Zhihui Zhang / Kai Jiang / Yan Xue / Yong Li / Peiyi Wang / Jixian Zhai / Hongwei Guo / Jiamu Du /
Abstract: Small RNAs, including microRNA, small interfering RNA (siRNA) and PIWI-interacting RNA, are regulatory RNAs that play critical roles in gene regulation, development, viral defence and environmental ...Small RNAs, including microRNA, small interfering RNA (siRNA) and PIWI-interacting RNA, are regulatory RNAs that play critical roles in gene regulation, development, viral defence and environmental response. The biogenesis of microRNA and siRNA relies on the Dicer family ribonucleases to capture, measure and cleave their double-stranded RNA substrates. In Arabidopsis, DICER-LIKE 4 (DCL4) produces 21-nucleotide siRNA in association with Double-Stranded RNA-Binding Protein 4 (DRB4) for post-transcriptional gene silencing. Here we determined the structures of the DCL4-RNA complex in a dicing-competent conformation and the DCL4-DRB4-RNA complex in a pre-dicing conformation. DCL4 measures 21 nucleotides along RNA between its PAZ and RNase III domains to determine the product siRNA length. A DCL4-specific loop locates the second double-stranded RNA binding domain of DCL4 and DRB4 to a distal position of the substrate RNA, yielding a preference for long RNA substrates. Our studies demonstrate the molecular basis of substrate recognition, length measurement and long RNA preference by the DCL4-DRB4 complex for 21-nucleotide siRNA biogenesis in plants.
History
DepositionOct 31, 2024-
Header (metadata) releaseFeb 18, 2026-
Map releaseFeb 18, 2026-
UpdateApr 15, 2026-
Current statusApr 15, 2026Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_62234.png

Downloads & links

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Map

FileDownload / File: emd_62234.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 360 pix.
= 307.8 Å		0.86 Å/pix.
x 360 pix.
= 307.8 Å		0.86 Å/pix.
x 360 pix.
= 307.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.855 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.0
Minimum - Maximum-0.15005371 - 18.383185999999998
Average (Standard dev.)-0.06336363 (±0.35396144)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 307.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_62234_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_62234_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : DCL4-RNA

EntireName: DCL4-RNA
Components
  • Complex: DCL4-RNA
    • Protein or peptide: Dicer-like protein 4
    • RNA: RNA (70-MER)
  • Ligand: MAGNESIUM ION

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Supramolecule #1: DCL4-RNA

SupramoleculeName: DCL4-RNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 210 KDa

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Macromolecule #1: Dicer-like protein 4

MacromoleculeName: Dicer-like protein 4 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on ester bonds; Endoribonucleases producing 5'-phosphomonoesters
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 193.967438 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MWSHPQFEKG SGSGSGSWSH PQFEKSYYHH HHHHHHHHDY DIPTTENLYF QGAMPYSEIG DDLRSLDFDH ADVSSDLHLT SSSSVSSFS SSSSSLFSAA GTDDPSPKME KDPRKIARRY QVELCKKATE ENVIVYLGTG CGKTHIAVML IYELGHLVLS P KKSVCIFL ...String:
MWSHPQFEKG SGSGSGSWSH PQFEKSYYHH HHHHHHHHDY DIPTTENLYF QGAMPYSEIG DDLRSLDFDH ADVSSDLHLT SSSSVSSFS SSSSSLFSAA GTDDPSPKME KDPRKIARRY QVELCKKATE ENVIVYLGTG CGKTHIAVML IYELGHLVLS P KKSVCIFL APTVALVEQQ AKVIADSVNF KVAIHCGGKR IVKSHSEWER EIAANEVLVM TPQILLHNLQ HCFIKMECIS LL IFDECHH AQQQSNHPYA EIMKVFYKSE SLQRPRIFGM TASPVVGKGS FQSENLSKSI NSLENLLNAK VYSVESNVQL DGF VSSPLV KVYYYRSALS DASQSTIRYE NMLEDIKQRC LASLKLLIDT HQTQTLLSMK RLLKRSHDNL IYTLLNLGLW GAIQ AAKIQ LNSDHNVQDE PVGKNPKSKI CDTYLSMAAE ALSSGVAKDE NASDLLSLAA LKEPLFSRKL VQLIKILSVF RLEPH MKCI IFVNRIVTAR TLSCILNNLE LLRSWKSDFL VGLSSGLKSM SRRSMETILK RFQSKELNLL VATKVGEEGL DIQTCC LVI RYDLPETVTS FIQSRGRARM PQSEYAFLVD SGNEKEMDLI ENFKVNEDRM NLEITYRSSE ETCPRLDEEL YKVHETG AC ISGGSSISLL YKYCSRLPHD EFFQPKPEFQ FKPVDEFGGT ICRITLPANA PISEIESSLL PSTEAAKKDA CLKAVHEL H NLGVLNDFLL PDSKDEIEDE LSDDEFDFDN IKGEGCSRGD LYEMRVPVLF KQKWDPSTSC VNLHSYYIMF VPHPADRIY KKFGFFMKSP LPVEAETMDI DLHLAHQRSV SVKIFPSGVT EFDNDEIRLA ELFQEIALKV LFERGELIPD FVPLELQDSS RTSKSTFYL LLPLCLHDGE SVISVDWVTI RNCLSSPIFK TPSVLVEDIF PPSGSHLKLA NGCWNIDDVK NSLVFTTYSK Q FYFVADIC HGRNGFSPVK ESSTKSHVES IYKLYGVELK HPAQPLLRVK PLCHVRNLLH NRMQTNLEPQ ELDEYFIEIP PE LSHLKIK GLSKDIGSSL SLLPSIMHRM ENLLVAIELK HVLSASIPEI AEVSGHRVLE ALTTEKCHER LSLERLEVLG DAF LKFAVS RHLFLHHDSL DEGELTRRRS NVVNNSNLCR LAIKKNLQVY IRDQALDPTQ FFAFGHPCRV TCDEVASKEV HSLN RDLGI LESNTGEIRC SKGHHWLYKK TIANVVQALV GAFLVDSGFK GAVKFLKWIG VNVDFESLQV QDACIASRRY LPLTT RNNL ETLENQLDYK FLHKGLLVQA FIHPSYNRHG GGCYQRLEFL GDAVLDYLMT SYFFTVFPKL KPGQLTDLRS LSVNNE ALA NVAVSFSLKR FLFCESIYLH EVIEDYTNFL ASSPLASGQS EGPRCPKVLG NLVQSCLGAL FLDCGFNLNH VWTMMLS FL DPVKNLSNLQ ISPIKELIEL CQSYKWDREI SATKKDGAFT VELKVTKNGC CLTVSATGRN KREGTKKAAQ LMITNLKA H ENITTSHPLE DVLKNGIRNE AKLIGYNEDP IDVVDLVGLD VENLNILETF GGNSERSSSY VIRRGLPQAP SKTEDRLPQ KAIIKAGGPS SKTAKSLLHE TCVANCWKPP HFECCEEEGP GHLKSFVYKV ILEVEDAPNM TLECYGEARA TKKGAAEHAA QAAIWCLKH SGFLCDYKDD DDKGDYKDDD DKIDYKDDDD K

UniProtKB: Dicer-like protein 4

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Macromolecule #2: RNA (70-MER)

MacromoleculeName: RNA (70-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 22.440297 KDa
SequenceString:
GAGACUUGGG CAAUGUGACU GCUGAGGUUC GCAUAUGCGA ACCUCAGCAG UCACAUUGCC CAAGUCUCUU

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration0.2 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.15 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.5.3) / Number images used: 157005
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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