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- PDB-8kfp: Solution structure of Drosophila melanogaster R2D2 dsRBD1 -

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Basic information

Entry
Database: PDB / ID: 8kfp
TitleSolution structure of Drosophila melanogaster R2D2 dsRBD1
ComponentsR2D2
KeywordsRNA BINDING PROTEIN / RNAi / siRNA / Dicer-2 / dsRNA
Function / homology
Function and homology information


follicle cell of egg chamber stalk formation / regulatory ncRNA processing / regulation of regulatory ncRNA processing / RISC-loading complex / RISC complex assembly / siRNA binding / siRNA processing / RISC complex / double-stranded RNA binding / defense response to virus ...follicle cell of egg chamber stalk formation / regulatory ncRNA processing / regulation of regulatory ncRNA processing / RISC-loading complex / RISC complex assembly / siRNA binding / siRNA processing / RISC complex / double-stranded RNA binding / defense response to virus / nucleus / cytoplasm
Similarity search - Function
Double-stranded RNA binding motif / Double-stranded RNA binding motif / Double stranded RNA-binding domain (dsRBD) profile. / Double-stranded RNA-binding domain
Similarity search - Domain/homology
Biological speciesDrosophila melanogaster (fruit fly)
MethodSOLUTION NMR / molecular dynamics
Model detailsMODEL GENERATED BY ROSETTA VERSION 2021.16+release.8ee4f02
AuthorsAute, R. / Deshmukh, M.V.
Funding support India, 1items
OrganizationGrant numberCountry
Council of Scientific & Industrial Research (CSIR)MLP161 India
CitationJournal: Biophys.Chem. / Year: 2024
Title: Key arginine residues in R2D2 dsRBD1 and dsRBD2 lead the siRNA recognition in Drosophila melanogaster RNAi pathway.
Authors: Aute, R. / Waghela, N. / Deshmukh, M.V.
History
DepositionAug 16, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 26, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: R2D2


Theoretical massNumber of molelcules
Total (without water)11,3121
Polymers11,3121
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: NMR Distance Restraints, not applicable, gel filtration, Elutes as a monomer as evaluated by SEC, isothermal titration calorimetry, binds to canonical dsRNA substrates, NMR relaxation ...Evidence: NMR Distance Restraints, not applicable, gel filtration, Elutes as a monomer as evaluated by SEC, isothermal titration calorimetry, binds to canonical dsRNA substrates, NMR relaxation study, Rotational correlation time and the order parameter suggest that R2D2 dsRBD1 is a globular protein corresponding to a monomeric state.
TypeNameSymmetry operationNumber
identity operation1_5551
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)10 / 5000structures with the lowest energy
RepresentativeModel #1lowest energy

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Components

#1: Protein R2D2


Mass: 11311.823 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: dsRBD1 of R2D2 / Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: r2d2 / Plasmid: pET30a / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: Q2Q0L0

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic12D 1H-15N HSQC
122isotropic12D 1H-13C HSQC
132isotropic13D HNCO
152isotropic13D HN(CA)CB
162isotropic13D HN(CA)CO
172isotropic13D HN(COCA)CB
182isotropic13D H(CCO)NH TOCSY
192isotropic13D C(CO)NH TOCSY
1102isotropic13D 1H-15N NOESY
1112isotropic13D 1H-13C NOESY

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Sample preparation

Details
TypeSolution-IDContentsDetailsLabelSolvent system
solution1500 uM [U-99% 15N] R2D2 dsRBD1, 90% H2O/10% D2O50 mM Kphosphate (pH 7.0), 50 mM Na2SO4, 50 mM NaCl, and 2 mM DTT15N90% H2O/10% D2O
solution2500 uM [U-99% 13C; U-99% 15N] R2D2 dsRBD1, 90% H2O/10% D2O50 mM Kphosphate (pH 7.0), 50 mM Na2SO4, 50 mM NaCl, and 2 mM DTT13C, 15N90% H2O/10% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
500 uMR2D2 dsRBD1[U-99% 15N]1
500 uMR2D2 dsRBD1[U-99% 13C; U-99% 15N]2
Sample conditionsIonic strength: 150 mM / Label: conditions _1 / pH: 7.0 / Pressure: 1 atm / Temperature: 298 K

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NMR measurement

NMR spectrometerType: Bruker AVANCE NEO / Manufacturer: Bruker / Model: AVANCE NEO / Field strength: 600 MHz

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Processing

NMR software
NameVersionDeveloperClassification
CARA1.8.4Keller and Wuthrichchemical shift assignment
Rosetta2021.16.61629Raman, S., Lange, O. F., Rossi, P., Tyka, M., Wang, X., Aramini, J., ... & Baker, D. (2010). NMR structure determination for larger proteins using backbone-only data. Science, 327(5968), 1014-1018.structure calculation
CARA1.8.4Keller and Wuthrichpeak picking
TopSpin2.1Bruker Biospincollection
TopSpin4.0.6Bruker Biospinprocessing
RefinementMethod: molecular dynamics / Software ordinal: 2
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 5000 / Conformers submitted total number: 10

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