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- PDB-7vg2: Cryo-EM structure of Arabidopsis DCL3 in complex with a 40-bp RNA -

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Basic information

Entry
Database: PDB / ID: 7vg2
TitleCryo-EM structure of Arabidopsis DCL3 in complex with a 40-bp RNA
Components
  • Dicer-like 3
  • TAS1a forward strand (5'-phosphorylation)
  • TAS1a reverse strand
KeywordsRNA BINDING PROTEIN/RNA / DCL3 / RNA / RdDM / Dicer / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


ribonuclease III activity / regulatory ncRNA-mediated gene silencing / RNA processing / RNA binding / ATP binding
Similarity search - Function
Ribonuclease III family signature. / Ribonuclease III domain / Ribonuclease III family domain profile. / Ribonuclease III family / Ribonuclease III domain / PAZ domain superfamily / PAZ / PAZ domain / PAZ domain profile. / PAZ domain ...Ribonuclease III family signature. / Ribonuclease III domain / Ribonuclease III family domain profile. / Ribonuclease III family / Ribonuclease III domain / PAZ domain superfamily / PAZ / PAZ domain / PAZ domain profile. / PAZ domain / Ribonuclease III, endonuclease domain superfamily / DEAD/DEAH box helicase domain / DEAD/DEAH box helicase / Helicase conserved 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 3
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsWang, Q. / Du, J.
CitationJournal: Science / Year: 2021
Title: Mechanism of siRNA production by a plant Dicer-RNA complex in dicing-competent conformation.
Authors: Qian Wang / Yan Xue / Laixing Zhang / Zhenhui Zhong / Suhua Feng / Changshi Wang / Lifan Xiao / Zhenlin Yang / C Jake Harris / Zhe Wu / Jixian Zhai / Maojun Yang / Sisi Li / Steven E Jacobsen / Jiamu Du /
Abstract: In eukaryotes, small RNAs (sRNAs) play critical roles in multiple biological processes. Dicer endonucleases are a central part of sRNA biogenesis. In plants, DICER-LIKE PROTEIN 3 (DCL3) produces 24- ...In eukaryotes, small RNAs (sRNAs) play critical roles in multiple biological processes. Dicer endonucleases are a central part of sRNA biogenesis. In plants, DICER-LIKE PROTEIN 3 (DCL3) produces 24-nucleotide (nt) small interfering RNAs (siRNAs) that determine the specificity of the RNA-directed DNA methylation pathway. Here, we determined the structure of a DCL3–pre-siRNA complex in an active dicing-competent state. The 5′-phosphorylated A1 of the guide strand and the 1-nt 3′ overhang of the complementary strand are specifically recognized by a positively charged pocket and an aromatic cap, respectively. The 24-nt siRNA length dependence relies on the separation between the 5′-phosphorylated end of the guide RNA and dual cleavage sites formed by the paired ribonuclease III domains. These structural studies, complemented by functional data, provide insight into the dicing principle for Dicers in general.
History
DepositionSep 14, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Oct 27, 2021Provider: repository / Type: Initial release
Revision 1.1Dec 15, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Jun 19, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

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Assembly

Deposited unit
A: Dicer-like 3
C: TAS1a forward strand (5'-phosphorylation)
D: TAS1a reverse strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)208,1786
Polymers208,0323
Non-polymers1463
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Dicer-like 3


Mass: 182176.766 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: DCL3, ATDCL3, DICER-LIKE 3, dicer-like 3, At3g43920 / Plasmid: pFastHTB / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: F4J0I5
#2: RNA chain TAS1a forward strand (5'-phosphorylation)


Mass: 12812.680 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Arabidopsis thaliana (thale cress)
#3: RNA chain TAS1a reverse strand


Mass: 13042.688 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Arabidopsis thaliana (thale cress)
#4: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestY

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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

Component
IDNameTypeEntity IDParent-IDSource
1DCL3 in complex with a 40-bp RNACOMPLEX#1-#30MULTIPLE SOURCES
2DCL3COMPLEX#11RECOMBINANT
3RNACOMPLEX#2-#31SYNTHETIC
Molecular weightValue: 0.21 MDa / Experimental value: YES
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris hydrochlorideTris-HCl,1
2100 mMsodium chlorideNaCl1
32 mMcalcium chlorideCaCl21
41 mMDithiothreitolDTT1
SpecimenConc.: 1.8 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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameCategory
2SerialEMimage acquisition
4GctfCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 220948 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL

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