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- PDB-3hk2: Crystal structure of T. thermophilus Argonaute N478 mutant protei... -

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Basic information

Entry
Database: PDB / ID: 3hk2
TitleCrystal structure of T. thermophilus Argonaute N478 mutant protein complexed with DNA guide strand and 19-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*TP*AP*GP*T)-3'
  • 5'-R(*UP*AP*UP*AP*CP*AP*AP*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 / Argonaute, N-terminal domain / Argonaute, middle domain / paz domain ...paz domain - #50 / Alpha-D-Glucose-1,6-Bisphosphate; Chain A, domain 4 - #60 / Alpha-Beta Plaits - #2620 / Argonaute, PAZ domain / : / : / Argonaute PAZ domain / Argonaute, N-terminal domain / Argonaute, middle domain / paz domain / Piwi domain profile. / Piwi domain / Piwi domain / Piwi / PAZ domain profile. / PAZ domain / Alpha-D-Glucose-1,6-Bisphosphate; Chain A, domain 4 / Response regulator / Ribonuclease H-like superfamily/Ribonuclease H / Beta Complex / 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: 2.8 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
DepositionMay 22, 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 ...chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model / struct_ncs_dom_lim
Item: _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id ..._struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Argonaute
B: Argonaute
C: 5'-D(P*TP*GP*AP*GP*GP*TP*AP*GP*TP*AP*GP*GP*TP*TP*GP*TP*AP*TP*AP*GP*T)-3'
E: 5'-D(P*TP*GP*AP*GP*GP*TP*AP*GP*TP*AP*GP*GP*TP*TP*GP*TP*AP*TP*AP*GP*T)-3'
F: 5'-R(*UP*AP*UP*AP*CP*AP*AP*CP*CP*UP*AP*CP*UP*AP*CP*CP*UP*CP*G)-3'
D: 5'-R(*UP*AP*UP*AP*CP*AP*AP*CP*CP*UP*AP*CP*UP*AP*CP*CP*UP*CP*G)-3'
hetero molecules


Theoretical massNumber of molelcules
Total (without water)178,61410
Polymers178,5176
Non-polymers974
Water25214
1
A: Argonaute
C: 5'-D(P*TP*GP*AP*GP*GP*TP*AP*GP*TP*AP*GP*GP*TP*TP*GP*TP*AP*TP*AP*GP*T)-3'
D: 5'-R(*UP*AP*UP*AP*CP*AP*AP*CP*CP*UP*AP*CP*UP*AP*CP*CP*UP*CP*G)-3'
hetero molecules


Theoretical massNumber of molelcules
Total (without water)89,3075
Polymers89,2593
Non-polymers492
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6270 Å2
ΔGint-64 kcal/mol
Surface area31880 Å2
MethodPISA
2
B: Argonaute
E: 5'-D(P*TP*GP*AP*GP*GP*TP*AP*GP*TP*AP*GP*GP*TP*TP*GP*TP*AP*TP*AP*GP*T)-3'
F: 5'-R(*UP*AP*UP*AP*CP*AP*AP*CP*CP*UP*AP*CP*UP*AP*CP*CP*UP*CP*G)-3'
hetero molecules


Theoretical massNumber of molelcules
Total (without water)89,3075
Polymers89,2593
Non-polymers492
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area6300 Å2
ΔGint-64 kcal/mol
Surface area31990 Å2
MethodPISA
Unit cell
Length a, b, c (Å)111.193, 110.884, 174.339
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B

NCS domain segments:

Ens-ID: 1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11LEULEUGLUGLUchain A and (resseq 4:40 or resseq 42:83 or resseq...AA4 - 404 - 40
12VALVALGLYGLYchain A and (resseq 4:40 or resseq 42:83 or resseq...AA42 - 8342 - 83
13THRTHRLEULEUchain A and (resseq 4:40 or resseq 42:83 or resseq...AA85 - 21585 - 215
14SERSERALAALAchain A and (resseq 4:40 or resseq 42:83 or resseq...AA222 - 242222 - 242
15VALVALGLUGLUchain A and (resseq 4:40 or resseq 42:83 or resseq...AA244 - 268244 - 268
16LEULEULEULEUchain A and (resseq 4:40 or resseq 42:83 or resseq...AA281 - 366281 - 366
17ALAALALEULEUchain A and (resseq 4:40 or resseq 42:83 or resseq...AA368 - 465368 - 465
18GLYGLYTHRTHRchain A and (resseq 4:40 or resseq 42:83 or resseq...AA467 - 605467 - 605
19ARGARGVALVALchain A and (resseq 4:40 or resseq 42:83 or resseq...AA611 - 685611 - 685
21LEULEUGLUGLUchain B and (resseq 4:40 or resseq 42:83 or resseq...BB4 - 404 - 40
22VALVALGLYGLYchain B and (resseq 4:40 or resseq 42:83 or resseq...BB42 - 8342 - 83
23THRTHRLEULEUchain B and (resseq 4:40 or resseq 42:83 or resseq...BB85 - 21585 - 215
24SERSERALAALAchain B and (resseq 4:40 or resseq 42:83 or resseq...BB222 - 242222 - 242
25VALVALGLUGLUchain B and (resseq 4:40 or resseq 42:83 or resseq...BB244 - 268244 - 268
26LEULEULEULEUchain B and (resseq 4:40 or resseq 42:83 or resseq...BB281 - 366281 - 366
27ALAALALEULEUchain B and (resseq 4:40 or resseq 42:83 or resseq...BB368 - 465368 - 465
28GLYGLYTHRTHRchain B and (resseq 4:40 or resseq 42:83 or resseq...BB467 - 605467 - 605
29ARGARGVALVALchain B and (resseq 4:40 or resseq 42:83 or resseq...BB611 - 685611 - 685

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Components

#1: Protein Argonaute


Mass: 76727.750 Da / Num. of mol.: 2 / Mutation: D478N
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*TP*AP*GP*T)-3'


Mass: 6588.266 Da / Num. of mol.: 2 / Source method: obtained synthetically
#3: RNA chain 5'-R(*UP*AP*UP*AP*CP*AP*AP*CP*CP*UP*AP*CP*UP*AP*CP*CP*UP*CP*G)-3'


Mass: 5942.584 Da / Num. of mol.: 2 / Source method: obtained synthetically
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 14 / 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: 3.01 Å3/Da / Density % sol: 59.14 %
Crystal growTemperature: 308 K / Method: vapor diffusion, hanging drop / pH: 7
Details: 1.0 M succinic acid, 0.1 M Hepes, pH 7.0, 1% (w/v) polyethylene glycol monomethyl ether 2,000, VAPOR DIFFUSION, HANGING DROP, temperature 308 K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.97 Å
DetectorType: MARMOSAIC 300 mm CCD / Detector: CCD / Date: Mar 17, 2009
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97 Å / Relative weight: 1
ReflectionResolution: 2.8→50 Å / Num. obs: 53789 / % possible obs: 99.9 % / Observed criterion σ(F): -1 / Redundancy: 6.6 % / Biso Wilson estimate: 71.24 Å2 / Rmerge(I) obs: 0.056 / Net I/σ(I): 30.6
Reflection shellResolution: 2.8→2.9 Å / Redundancy: 6.3 % / Rmerge(I) obs: 0.487 / Mean I/σ(I) obs: 2.1 / Num. unique all: 5282 / % possible all: 99.9

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Processing

Software
NameVersionClassificationNB
PHENIXrefinement
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: 2.8→46.781 Å / Occupancy max: 1 / Occupancy min: 1 / FOM work R set: 0.82 / SU ML: 0.36 / Isotropic thermal model: Overall / Cross valid method: THROUGHOUT / σ(F): 0.07 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.241 2548 5.08 %RANDOM
Rwork0.205 ---
obs0.207 50206 93.26 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL / Bsol: 51.808 Å2 / ksol: 0.305 e/Å3
Displacement parametersBiso max: 189.5 Å2 / Biso mean: 79.639 Å2 / Biso min: 25.75 Å2
Baniso -1Baniso -2Baniso -3
1-5.788 Å20 Å2-0 Å2
2--6.359 Å20 Å2
3----12.147 Å2
Refinement stepCycle: LAST / Resolution: 2.8→46.781 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9770 1304 4 14 11092
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00711465
X-RAY DIFFRACTIONf_angle_d1.24215880
X-RAY DIFFRACTIONf_chiral_restr0.0751817
X-RAY DIFFRACTIONf_plane_restr0.0061861
X-RAY DIFFRACTIONf_dihedral_angle_d19.2854246
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDTypeRms dev position (Å)
11A4818X-RAY DIFFRACTIONPOSITIONAL0.024
12B4818X-RAY DIFFRACTIONPOSITIONAL0.024
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 18

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.8-2.8520.3291240.2922211233579
2.852-2.9110.3191180.292286240482
2.911-2.9740.2781250.2712354247985
2.974-3.0430.3151060.2892431253786
3.043-3.1190.3051300.2632519264990
3.119-3.2040.3111480.2482537268591
3.204-3.2980.3041540.2352620277493
3.298-3.4040.291510.2352670282195
3.404-3.5260.2511310.2122673280495
3.526-3.6670.2631360.2072730286696
3.667-3.8340.241550.1972720287596
3.834-4.0360.2151450.1772730287597
4.036-4.2880.1881530.1592790294398
4.288-4.6190.1741360.1462828296498
4.619-5.0840.1831500.1572829297999
5.084-5.8180.2291520.1812848300099
5.818-7.3260.2371530.20529003053100
7.326-46.7880.2251810.2072982316399

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