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- PDB-9kbz: Cryo-EM structure of DCL4-DRB4CTD-dsRNA complex -

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Basic information

Entry
Database: PDB / ID: 9kbz
TitleCryo-EM structure of DCL4-DRB4CTD-dsRNA complex
Components
  • Dicer-like protein 4
  • Double-stranded RNA-binding protein 4
  • RNA (55-MER)
  • RNA (57-MER)
KeywordsRNA BINDING PROTEIN/RNA / DCL4 / DRB4 / siRNA / Dicer / 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 / miRNA processing / siRNA processing / negative regulation of gene expression via chromosomal CpG island methylation ...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 / miRNA processing / siRNA processing / negative regulation of gene expression via chromosomal CpG island methylation / helicase activity / DNA-templated transcription termination / cytoplasmic ribonucleoprotein granule / double-stranded RNA binding / 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 / AtDRB-like, first double-stranded RNA binding domain, plant / AtDRB-like, second double-stranded RNA binding domain, plant / 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. ...double strand RNA binding domain from DEAD END PROTEIN 1 / AtDRB-like, first double-stranded RNA binding domain, plant / AtDRB-like, second double-stranded RNA binding domain, plant / 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 / Double-stranded RNA binding motif / 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
: / RNA / RNA (> 10) / Dicer-like protein 4 / Double-stranded RNA-binding protein 4
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.18 Å
AuthorsWang, C. / Du, J.
Funding support China, 1items
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, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Feb 18, 2026Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Dicer-like protein 4
B: Double-stranded RNA-binding protein 4
C: RNA (57-MER)
D: RNA (55-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)243,4277
Polymers243,3544
Non-polymers733
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Dicer-like protein 4


Mass: 193967.438 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: DCL4, At5g20320, F5O24.210 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P84634, Hydrolases; Acting on ester bonds; Endoribonucleases producing 5'-phosphomonoesters
#2: Protein Double-stranded RNA-binding protein 4 / dsRNA-binding protein 4 / AtDRB4


Mass: 13526.003 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: DBR4, At3g62800, F26K9.230 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q8H1D4
#3: RNA chain RNA (57-MER)


Mass: 18249.770 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Arabidopsis thaliana (thale cress) / References: GenBank: 20197647
#4: RNA chain RNA (55-MER) / RNA (5'-phosphorylated 55 MER)


Mass: 17610.512 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Arabidopsis thaliana (thale cress) / References: GenBank: 20197647
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Cryo-EM structure of DCL4 in complex with DRB4 CTD and RNA
Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.24 MDa / Experimental value: NO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenConc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.18 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 105170 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00514349
ELECTRON MICROSCOPYf_angle_d0.58219893
ELECTRON MICROSCOPYf_dihedral_angle_d7.2362795
ELECTRON MICROSCOPYf_chiral_restr0.0372355
ELECTRON MICROSCOPYf_plane_restr0.0052143

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