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- PDB-3ho1: Crystal structure of T. thermophilus Argonaute N546 mutant protei... -

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Basic information

Entry
Database: PDB / ID: 3ho1
TitleCrystal structure of T. thermophilus Argonaute N546 mutant protein complexed with DNA guide strand and 12-nt RNA target strand
Components
  • 5'-D(P*TP*GP*AP*GP*GP*TP*AP*GP*TP*AP*GP*GP*TP*TP*GP*TP*AP*T*AP*GP*T)-3'
  • 5'-R(P*CP*CP*UP*AP*CP*UP*AP*CP*CP*UP*CP*G)-3'
  • argonaute
KeywordsNUCLEIC ACID BINDING PROTEIN/DNA/RNA / argonaute / protein-DNA-RNA complex / NUCLEIC ACID BINDING PROTEIN-DNA-RNA COMPLEX
Function / homology
Function and homology information


Hydrolases; Acting on ester bonds; Site specific endodeoxyribonucleases: cleavage is not sequence specific (deleted sub-subclass) / clearance of foreign intracellular DNA / DNA endonuclease activity / manganese ion binding / DNA replication / DNA binding / RNA binding
Similarity search - Function
paz domain - #50 / Alpha-D-Glucose-1,6-Bisphosphate; Chain A, domain 4 - #60 / Alpha-Beta Plaits - #2620 / Argonaute, PAZ domain / Argonaute PAZ domain / paz domain / Piwi domain / Piwi domain profile. / Piwi domain / Piwi ...paz domain - #50 / Alpha-D-Glucose-1,6-Bisphosphate; Chain A, domain 4 - #60 / Alpha-Beta Plaits - #2620 / Argonaute, PAZ domain / Argonaute PAZ domain / paz domain / Piwi domain / Piwi domain profile. / Piwi domain / Piwi / PAZ domain profile. / PAZ domain / Alpha-D-Glucose-1,6-Bisphosphate; Chain A, domain 4 / Response regulator / Beta Complex / Ribonuclease H-like superfamily/Ribonuclease H / Nucleotidyltransferase; domain 5 / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Alpha-Beta Plaits / Rossmann fold / 2-Layer Sandwich / 3-Layer(aba) Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / Protein argonaute
Similarity search - Component
Biological speciesThermus thermophilus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.6 Å
AuthorsWang, Y. / Li, H. / Sheng, G. / Patel, D.J.
CitationJournal: Nature / Year: 2009
Title: Nucleation, propagation and cleavage of target RNAs in Ago silencing complexes.
Authors: Wang, Y. / Juranek, S. / Li, H. / Sheng, G. / Wardle, G.S. / Tuschl, T. / Patel, D.J.
History
DepositionJun 1, 2009Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 6, 2009Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Source and taxonomy / Version format compliance
Revision 1.2Oct 13, 2021Group: Database references / Derived calculations / Category: database_2 / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.3Sep 6, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: argonaute
X: 5'-D(P*TP*GP*AP*GP*GP*TP*AP*GP*TP*AP*GP*GP*TP*TP*GP*TP*AP*T*AP*GP*T)-3'
Y: 5'-R(P*CP*CP*UP*AP*CP*UP*AP*CP*CP*UP*CP*G)-3'
hetero molecules


Theoretical massNumber of molelcules
Total (without water)87,0494
Polymers87,0243
Non-polymers241
Water1,67593
1


  • Idetical with deposited unit
  • defined by software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5830 Å2
ΔGint-52 kcal/mol
Surface area31650 Å2
MethodPISA
Unit cell
Length a, b, c (Å)122.928, 118.531, 59.533
Angle α, β, γ (deg.)90.000, 114.910, 90.000
Int Tables number5
Space group name H-MC121

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Components

#1: Protein argonaute


Mass: 76727.750 Da / Num. of mol.: 1 / Mutation: D546N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus thermophilus (bacteria) / Strain: HB27 / Gene: TT_P0026 / Plasmid: PET-Sumo / Production host: Escherichia coli (E. coli) / Strain (production host): ROSETTA 2 (DE3)pLysS / References: UniProt: Q746M7
#2: DNA chain 5'-D(P*TP*GP*AP*GP*GP*TP*AP*GP*TP*AP*GP*GP*TP*TP*GP*TP*AP*T*AP*GP*T)-3'


Mass: 6588.266 Da / Num. of mol.: 1 / Source method: obtained synthetically
#3: RNA chain 5'-R(P*CP*CP*UP*AP*CP*UP*AP*CP*CP*UP*CP*G)-3'


Mass: 3708.248 Da / Num. of mol.: 1 / Source method: obtained synthetically
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 93 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.26 Å3/Da / Density % sol: 45.59 %
Crystal growTemperature: 308 K / Method: vapor diffusion, sitting drop / pH: 6
Details: containing 2.5 mM spermine, 10 mM MgCl2, 5 mM CaCl2, 50 mM Na cacodylate pH 6.0, 10% (v/v) isopropanol, VAPOR DIFFUSION, SITTING DROP, temperature 308 K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.979 Å
DetectorType: MARMOSAIC 300 mm CCD / Detector: CCD / Date: Dec 15, 2008
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979 Å / Relative weight: 1
ReflectionResolution: 2.6→50 Å / Num. obs: 23588 / % possible obs: 99.9 % / Observed criterion σ(F): -3 / Redundancy: 5.5 % / Rmerge(I) obs: 0.075 / Net I/σ(I): 26.4
Reflection shellResolution: 2.6→2.64 Å / Redundancy: 5.4 % / Rmerge(I) obs: 0.48 / Mean I/σ(I) obs: 2.8 / Num. unique all: 1155 / % possible all: 99.8

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Processing

Software
NameVersionClassificationNB
CNSrefinement
PDB_EXTRACT3.005data extraction
HKL-2000data collection
HKL-2000data reduction
HKL-2000data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 3DLH

3dlh


Resolution: 2.6→30 Å / Occupancy max: 1 / Occupancy min: 0.5 / Isotropic thermal model: Isotropic / Cross valid method: THROUGHOUT / σ(F): 0 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.258 1546 6.5 %RANDOM
Rwork0.22 ---
obs-22630 95 %-
Solvent computationBsol: 36.035 Å2
Displacement parametersBiso max: 134.85 Å2 / Biso mean: 61.367 Å2 / Biso min: 22.57 Å2
Baniso -1Baniso -2Baniso -3
1-5.499 Å20 Å214.328 Å2
2--5.337 Å20 Å2
3----10.836 Å2
Refinement stepCycle: LAST / Resolution: 2.6→30 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5145 528 1 93 5767
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_bond_d0.009
X-RAY DIFFRACTIONc_angle_d1.796
Xplor file
Refine-IDSerial noParam file
X-RAY DIFFRACTION1CNS_TOPPAR:protein_rep.param
X-RAY DIFFRACTION2CNS_TOPPAR:dna-rna_rep.param
X-RAY DIFFRACTION3CNS_TOPPAR:ion.param
X-RAY DIFFRACTION4CNS_TOPPAR:water_rep.param

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