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- PDB-9mw8: Cryo-EM structure of ancestral Dicer helicase bound to 27-bp dsRN... -

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Basic information

Entry
Database: PDB / ID: 9mw8
TitleCryo-EM structure of ancestral Dicer helicase bound to 27-bp dsRNA in post-hydrolysis closed state
Components
  • (RNA (27-MER)) x 2
  • AncD1D2
KeywordsANTIVIRAL PROTEIN/RNA / RNA helicase / dsRBM / ANTIVIRAL PROTEIN-RNA complex
Function / homologyADENOSINE-5'-DIPHOSPHATE / RNA / RNA (> 10)
Function and homology information
Biological speciessynthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsAderounmu, A.M. / Consalvo, C.D. / Shen, P.S. / Bass, B.L.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM141262 United States
Citation
Journal: Proc Natl Acad Sci U S A / Year: 2025
Title: Biochemical and structural basis of Dicer helicase function unveiled by resurrecting ancient proteins.
Authors: Adedeji M Aderounmu / Josephine Maus-Conn / Claudia D Consalvo / Peter S Shen / Brenda L Bass /
Abstract: A fully functional Dicer helicase, present in the modern arthropod, uses energy from ATP hydrolysis to power translocation on bound dsRNA, enabling the processive dsRNA cleavage required for ...A fully functional Dicer helicase, present in the modern arthropod, uses energy from ATP hydrolysis to power translocation on bound dsRNA, enabling the processive dsRNA cleavage required for efficient antiviral defense. However, modern Dicer orthologs exhibit divergent helicase functions that affect their ability to contribute to antiviral defense. Moreover, mechanisms that couple ATP hydrolysis to Dicer helicase movement on dsRNA remain enigmatic. We used biochemical and structural analyses of ancestrally reconstructed Dicer helicases to map evolution of dsRNA binding affinity, ATP hydrolysis and translocation. Loss of affinity for dsRNA occurred early in Dicer evolution, coinciding with a decline in translocation activity, despite preservation of ATP hydrolysis activity. Ancestral nematode Dicer also exhibited significant decline in ATP hydrolysis and translocation, but studies of antiviral activities in the modern nematode indicate Dicer retained a role in antiviral defense by recruiting a second helicase. Cryogenic electron microscopy (cryo-EM) analyses of an ancient metazoan Dicer allowed capture of multiple helicase states revealing the mechanism that connects each step of ATP hydrolysis to unidirectional movement along dsRNA. Our study rationalizes the diversity in modern Dicer helicases by connecting ancestral functions to observations in extant enzymes.
#1: Journal: bioRxiv / Year: 2025
Title: Biochemical and structural basis of Dicer helicase function unveiled by resurrecting ancient proteins.
Authors: Adedeji M Aderounmu / Josephine Maus-Conn / Claudia D Consalvo / Peter S Shen / Brenda L Bass /
Abstract: A fully functional Dicer helicase, present in the modern arthropod, uses energy generated during ATP hydrolysis to power translocation on bound dsRNA, enabling the processive dsRNA cleavage required ...A fully functional Dicer helicase, present in the modern arthropod, uses energy generated during ATP hydrolysis to power translocation on bound dsRNA, enabling the processive dsRNA cleavage required for efficient antiviral defense. However, modern Dicer orthologs exhibit divergent helicase functions that affect their ability to contribute to antiviral defense, and moreover, mechanisms that couple ATP hydrolysis to Dicer helicase movement on dsRNA remain enigmatic. Here, we used biochemical and structural analyses of ancestrally reconstructed Dicer helicases to map evolution of dsRNA binding affinity, ATP hydrolysis and translocation. We found that loss of affinity for dsRNA occurred early in Dicer evolution, coinciding with a decline in translocation activity, despite preservation of ATP hydrolysis activity, exemplified by the ancient deuterostome Dicer. Ancestral nematode Dicer also exhibited significant decline in ATP hydrolysis and translocation, but studies of antiviral activities in the modern nematode indicate Dicer retained a role in antiviral defense by recruiting a second helicase. Cryo-EM analyses of an ancient metazoan Dicer allowed capture of multiple helicase states revealing the mechanism that connects each step of ATP hydrolysis to unidirectional movement along dsRNA. Overall, our study rationalizes the diversity in modern Dicer helicases by connecting ancestral functions to observations in extant enzymes.
History
DepositionJan 17, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 12, 2025Provider: repository / Type: Initial release
Revision 1.1Jun 4, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: AncD1D2
B: RNA (27-MER)
C: RNA (27-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)93,1625
Polymers92,7113
Non-polymers4522
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein AncD1D2


Mass: 75480.609 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Spodoptera frugiperda (fall armyworm)
#2: RNA chain RNA (27-MER)


Mass: 8572.096 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: synthetic construct (others)
#3: RNA chain RNA (27-MER)


Mass: 8658.198 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: synthetic construct (others)
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
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: Ancestral metazoan Dicer helicase in complex 27-bp blunt dsRNA and ATP
Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.093 MDa / Experimental value: NO
Source (natural)Organism: synthetic construct (others)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen 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 magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2EPUimage acquisition
4cryoSPARCCTF correction
7Cootmodel fitting
9PHENIX1.21.1_5286model refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 315941 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0026640
ELECTRON MICROSCOPYf_angle_d0.6059206
ELECTRON MICROSCOPYf_dihedral_angle_d14.2011460
ELECTRON MICROSCOPYf_chiral_restr0.0361078
ELECTRON MICROSCOPYf_plane_restr0.004984

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