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- PDB-8dg5: Structural Basis of MicroRNA Biogenesis by Dicer-1 and Its Partne... -

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Basic information

Entry
Database: PDB / ID: 8dg5
TitleStructural Basis of MicroRNA Biogenesis by Dicer-1 and Its Partner Protein Loqs-PB - complex IIb
Components
  • Endoribonuclease Dcr-1
  • Loquacious, isoform B
  • RNA (60-MER)
KeywordsRNA BINDING PROTEIN/RNA / Dicer / Dcr-1 / Loquacious / Loqs-PB / miRNA / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


mitotic cell cycle, embryonic / germarium-derived female germ-line cyst formation / germarium-derived oocyte fate determination / germ-line stem cell division / pole cell formation / MicroRNA (miRNA) biogenesis / segment polarity determination / Small interfering RNA (siRNA) biogenesis / PKR-mediated signaling / female germ-line stem cell asymmetric division ...mitotic cell cycle, embryonic / germarium-derived female germ-line cyst formation / germarium-derived oocyte fate determination / germ-line stem cell division / pole cell formation / MicroRNA (miRNA) biogenesis / segment polarity determination / Small interfering RNA (siRNA) biogenesis / PKR-mediated signaling / female germ-line stem cell asymmetric division / regulation of regulatory ncRNA processing / dosage compensation by hyperactivation of X chromosome / RISC complex binding / pre-miRNA binding / apoptotic DNA fragmentation / germ-line stem cell population maintenance / ribonuclease III / deoxyribonuclease I activity / RISC-loading complex / miRNA metabolic process / RISC complex assembly / regulatory ncRNA-mediated post-transcriptional gene silencing / miRNA processing / ribonuclease III activity / pre-miRNA processing / siRNA processing / siRNA binding / RISC complex / response to starvation / dendrite morphogenesis / RNA endonuclease activity / central nervous system development / helicase activity / double-stranded RNA binding / single-stranded RNA binding / RNA binding / ATP binding / metal ion binding / nucleus / cytoplasm / cytosol
Similarity search - Function
Dicer, double-stranded RNA-binding domain / : / Dicer, partner-binding domain / Dicer, dsRNA-binding domain / : / : / Dicer, platform domain / Dicer dimerisation domain / Dicer dimerisation domain superfamily / Dicer dimerisation domain ...Dicer, double-stranded RNA-binding domain / : / Dicer, partner-binding domain / Dicer, dsRNA-binding domain / : / : / Dicer, platform domain / 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 domain superfamily / PAZ / PAZ domain / PAZ domain profile. / PAZ domain / Double-stranded RNA binding motif / Double-stranded RNA binding motif / Ribonuclease III, endonuclease domain superfamily / Double stranded RNA-binding domain (dsRBD) profile. / Double-stranded RNA-binding domain / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Helicase, C-terminal / P-loop containing nucleotide triphosphate hydrolases / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
URIDINE-5'-MONOPHOSPHATE / RNA / RNA (> 10) / Endoribonuclease Dcr-1 / Protein Loquacious
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.26 Å
AuthorsJouravleva, K. / Golovenko, D. / Demo, G. / Dutcher, R.C. / Tanaka Hall, T.M. / Zamore, P.D. / Korostelev, A.A.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM136275 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM127094 United States
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)1ZIA50165 United States
CitationJournal: Mol Cell / Year: 2022
Title: Structural basis of microRNA biogenesis by Dicer-1 and its partner protein Loqs-PB.
Authors: Karina Jouravleva / Dmitrij Golovenko / Gabriel Demo / Robert C Dutcher / Traci M Tanaka Hall / Phillip D Zamore / Andrei A Korostelev /
Abstract: In animals and plants, Dicer enzymes collaborate with double-stranded RNA-binding domain (dsRBD) proteins to convert precursor-microRNAs (pre-miRNAs) into miRNA duplexes. We report six cryo-EM ...In animals and plants, Dicer enzymes collaborate with double-stranded RNA-binding domain (dsRBD) proteins to convert precursor-microRNAs (pre-miRNAs) into miRNA duplexes. We report six cryo-EM structures of Drosophila Dicer-1 that show how Dicer-1 and its partner Loqs‑PB cooperate (1) before binding pre-miRNA, (2) after binding and in a catalytically competent state, (3) after nicking one arm of the pre-miRNA, and (4) following complete dicing and initial product release. Our reconstructions suggest that pre-miRNA binds a rare, open conformation of the Dicer‑1⋅Loqs‑PB heterodimer. The Dicer-1 dsRBD and three Loqs‑PB dsRBDs form a tight belt around the pre-miRNA, distorting the RNA helix to place the scissile phosphodiester bonds in the RNase III active sites. Pre-miRNA cleavage shifts the dsRBDs and partially closes Dicer-1, which may promote product release. Our data suggest a model for how the Dicer‑1⋅Loqs‑PB complex affects a complete cycle of pre-miRNA recognition, stepwise endonuclease cleavage, and product release.
History
DepositionJun 23, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 16, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 12, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Endoribonuclease Dcr-1
E: RNA (60-MER)
K: Loquacious, isoform B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)325,27115
Polymers324,6803
Non-polymers59212
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Endoribonuclease Dcr-1 / Protein dicer-1


Mass: 255733.797 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: Dcr-1, CG4792 / Production host: unidentified baculovirus
References: UniProt: Q9VCU9, Hydrolases; Acting on ester bonds; Endoribonucleases producing 5'-phosphomonoesters
#2: RNA chain RNA (60-MER)


Mass: 18796.139 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Drosophila melanogaster (fruit fly)
#3: Protein Loquacious, isoform B / R3D1-L / RE14437p


Mass: 50149.867 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: CG6866 / Production host: unidentified baculovirus / References: UniProt: Q9VJY9
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-U5P / URIDINE-5'-MONOPHOSPHATE


Mass: 324.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C9H13N2O9P
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Ternary complex of Dicer-1 and Loqs-PB binding pre-miRNA
Type: COMPLEX / Entity ID: #1, #3 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Source (recombinant)Organism: unidentified baculovirus
Buffer solutionpH: 7.9
Buffer componentConc.: 20 mM / Name: HEPES-KOH / Formula: HEPES-KOH
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 293 K

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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 / Nominal defocus max: 3500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 66.3 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameCategory
4CTFFINDCTF correction
10RELIONinitial Euler assignment
12RELIONclassification
13cryoSPARC3D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 3161540
3D reconstructionResolution: 3.26 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 185002 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 71.54 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01116718
ELECTRON MICROSCOPYf_angle_d1.403222885
ELECTRON MICROSCOPYf_chiral_restr0.06142595
ELECTRON MICROSCOPYf_plane_restr0.00942769
ELECTRON MICROSCOPYf_dihedral_angle_d9.66292670

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