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- PDB-2ltr: Solution structure of RDE-4(32-136) -

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Basic information

Entry
Database: PDB / ID: 2ltr
TitleSolution structure of RDE-4(32-136)
ComponentsProtein RDE-4
KeywordsRNA BINDING PROTEIN / RDE-4 / dsRBD1 / RNAi / RDE-4DC
Function / homology
Function and homology information


MicroRNA (miRNA) biogenesis / Small interfering RNA (siRNA) biogenesis / PKR-mediated signaling / regulation of regulatory ncRNA processing / RISC-loading complex / regulatory ncRNA-mediated post-transcriptional gene silencing / siRNA processing / siRNA binding / RISC complex / double-stranded RNA binding ...MicroRNA (miRNA) biogenesis / Small interfering RNA (siRNA) biogenesis / PKR-mediated signaling / regulation of regulatory ncRNA processing / RISC-loading complex / regulatory ncRNA-mediated post-transcriptional gene silencing / siRNA processing / siRNA binding / RISC complex / double-stranded RNA binding / single-stranded RNA binding / ribonucleoprotein complex / protein homodimerization activity / nucleus / cytoplasm
Similarity search - Function
: / Double Stranded RNA Binding Domain - #20 / Double-stranded RNA binding motif / Double-stranded RNA binding motif / Double stranded RNA-binding domain (dsRBD) profile. / Double-stranded RNA-binding domain / Double Stranded RNA Binding Domain / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
DRBM domain-containing protein
Similarity search - Component
Biological speciesCaenorhabditis elegans (invertebrata)
MethodSOLUTION NMR / simulated annealing
AuthorsDeshmukh, M. / Chiliveri, S.
Citation
Journal: Biochem.J. / Year: 2014
Title: Structure of RDE-4 dsRBDs and mutational studies provide insights into dsRNA recognition in the Caenorhabditis elegans RNAi pathway.
Authors: Chiliveri, S.C. / Deshmukh, M.V.
#1: Journal: Biomol.Nmr Assign. / Year: 2011
Title: Backbone and sidechain methyl Ile (delta1), Leu and Val chemical shift assignments of RDE-4 (1-243), an RNA interference initiation protein in C. elegans.
Authors: Chiliveri, S.C. / Kumar, S. / Marelli, U.K. / Deshmukh, M.V.
History
DepositionMay 31, 2012Deposition site: BMRB / Processing site: RCSB
Revision 1.0Dec 4, 2013Provider: repository / Type: Initial release
Revision 1.1Feb 12, 2014Group: Database references
Revision 1.2Apr 27, 2016Group: Structure summary
Revision 1.3Jun 14, 2023Group: Data collection / Database references / Other
Category: database_2 / pdbx_database_status ...database_2 / pdbx_database_status / pdbx_nmr_software / pdbx_nmr_spectrometer / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.status_code_nmr_data / _pdbx_nmr_software.name / _pdbx_nmr_spectrometer.model / _struct_ref_seq_dif.details
Revision 1.4May 15, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2 / Item: _database_2.pdbx_DOI

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Protein RDE-4


Theoretical massNumber of molelcules
Total (without water)27,8611
Polymers27,8611
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)10 / 100structures with the lowest energy
RepresentativeModel #1lowest energy

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Components

#1: Protein Protein RDE-4 / RNA interference promoting factor


Mass: 27860.629 Da / Num. of mol.: 1 / Fragment: dsRBD1 (UniProt residues 32-136)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caenorhabditis elegans (invertebrata) / Gene: rde-4, T20G5.11 / Plasmid: pET28a / Production host: Escherichia coli (E. coli) / References: UniProt: G5EBF5

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
Details: Solution structure of RDE-4(32-136) containing N-terminal helix, dsRBD1 and part of the linker
NMR experiment
Conditions-IDExperiment-IDSolution-IDType
1112D 1H-15N HSQC
1223D HNCO
1323D HNCA
1423D HN(COCA)CB
1523D HN(CO)CA
1623D HN(CA)CO
1723D HN(CA)CB
1833D H(CCCO)NH TOCSY
1933D (H)C(CCO)NH TOCSY
11013D 1H-15N NOESY
11143D 1H-13C(Me-Only) NOESY
11243D 1H-13C NOESY aromatic
11352D 1H-13C HSQC
11422D 1H-13C HSQC
11542D 1H-13C HSQC
21612D 1H-15N HSQC

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Sample preparation

Details
Solution-IDContentsSolvent system
1200 uM [U-100% 15N] RDE-4DC, 90% H2O/10% D2O90% H2O/10% D2O
2500 uM [U-100% 13C; U-100% 15N; U-80% 2H] RDE-4DC, 90% H2O/10% D2O90% H2O/10% D2O
3500 uM [U-100% 15N; U-100% 13C; U-100% 2H] RDE-4DC with [U-100% 1H] Val, Leu, and Ile RDE-4DC, 90% H2O/10% D2O90% H2O/10% D2O
4500 uM [U-100% 15N; U-100% 2H] RDE-4DC with [U-100% 13C-methyl] Val, Leu, and Ile (d1) and [U-100% 1H] Ile, Leu, Val and Phe RDE-4DC, 500 uM [U-100% 15N; U-100% 2H] RDE-4DC with [U-100% 13C-methyl] Val, Leu, and Ile (d1) and [U-100% 1H] Ile, Leu, Val and Tyr RDE-4DC, 90% H2O/10% D2O90% H2O/10% D2O
5200 uM [U-10% 13C] RDE-4DC, 90% H2O/10% D2O90% H2O/10% D2O
6300 uM RDE-4DC, 90% H2O/10% D2O90% H2O/10% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
200 uMRDE-4DC-1[U-100% 15N]1
500 uMRDE-4DC-2[U-100% 13C; U-100% 15N; U-80% 2H]2
500 uMRDE-4DC-3[U-100% 15N; U-100% 13C; U-100% 2H] RDE-4DC with [U-100% 1H] Val, Leu, and Ile3
500 uMRDE-4DC-4[U-100% 15N; U-100% 2H] RDE-4DC with [U-100% 13C-methyl] Val, Leu, and Ile (d1) and [U-100% 1H] Ile, Leu, Val and Phe4
500 uMRDE-4DC-5[U-100% 15N; U-100% 2H] RDE-4DC with [U-100% 13C-methyl] Val, Leu, and Ile (d1) and [U-100% 1H] Ile, Leu, Val and Tyr4
200 uMRDE-4DC-6[U-10% 13C]5
300 uMRDE-4DC-76
Sample conditions
Conditions-IDIonic strengthpHPressure (kPa)Temperature (K)
10.157ambient 298 K
20.37ambient 298 K

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NMR measurement

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

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Processing

NMR software
NameDeveloperClassification
X-PLOR NIHSchwieters, Kuszewski, Tjandra and Clorestructure solution
ProcheckNMRLaskowski and MacArthurstructure validation
TopSpinBruker Biospincollection
NMRViewJohnson, One Moon Scientificchemical shift assignment
CARAKeller and Wuethrichchemical shift assignment
X-PLOR NIHSchwieters, Kuszewski, Tjandra and Clorerefinement
RefinementMethod: simulated annealing / Software ordinal: 1
Details: THE PROTEIN USED IN THIS STUDY (UNP RESIDUES 1-243) IS A FRAGMENT OF RDE-4, AN RNAI INITIATOR PROTEIN IN C. ELEGANS. IT CONSISTS OF FOUR REGIONS, WHICH ARE THE N-TERMINAL REGION (1-43), ...Details: THE PROTEIN USED IN THIS STUDY (UNP RESIDUES 1-243) IS A FRAGMENT OF RDE-4, AN RNAI INITIATOR PROTEIN IN C. ELEGANS. IT CONSISTS OF FOUR REGIONS, WHICH ARE THE N-TERMINAL REGION (1-43), DSRBD1 (44-107), LINKER (108-168), AND DSRBD2 (169-235). IN THE COURSE OF ASSIGNMENTS AND STRUCTURE CALCULATION, A PART OF THE LINKER (RESIDUES 126-149) IS FOUND TO BE HIGHLY FLEXIBLE. NO SPATIAL INTER-DOMAIN CONTACTS WERE OBSERVED BETWEEN THE TWO DSRBDS. THEREFORE, SOLUTION STRUCTURES OF DSRBD1 AND DSRBD2 WERE CALCULATED SEPARATELY. FOR 2LTR, RIGID PART OF N-TERMINAL REGION (32-43), DSRBD1 (44-107) AND LINKER REGION (108-136) WERE USED TO CALCULATE THE FINAL STRUCTURES, WHEREAS FOR 2LTS, RIGID PART OF THE LINKER (150-168) AND DSRBD2 (169-235) WERE CHOSEN FOR THE CALCULATION OF THE FINAL STRUCTURES. THE RELATIVE ORIENTATION BETWEEN TWO STRUCTURES COULD NOT BE DETERMINED DUE TO THE FLEXIBLE NATURE OF THE LINKER.
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 100 / Conformers submitted total number: 10

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