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- PDB-5hnk: Crystal structure of T5Fen in complex intact substrate and metal ions. -

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Basic information

Entry
Database: PDB / ID: 5hnk
TitleCrystal structure of T5Fen in complex intact substrate and metal ions.
Components
  • DNA (5'-D(*AP*AP*AP*AP*GP*CP*GP*TP*AP*CP*GP*C)-3')
  • Exodeoxyribonuclease
KeywordsHYDROLASE / enzyme-DNA complex / flap endonuclease / metalloenzyme.
Function / homology
Function and homology information


viral replication complex / exodeoxyribonuclease (lambda-induced) / late viral transcription / DNA replication, Okazaki fragment processing / double-stranded DNA 5'-3' DNA exonuclease activity / double-stranded DNA endonuclease activity / DNA exonuclease activity / 5'-flap endonuclease activity / viral DNA genome replication / 5'-3' exonuclease activity ...viral replication complex / exodeoxyribonuclease (lambda-induced) / late viral transcription / DNA replication, Okazaki fragment processing / double-stranded DNA 5'-3' DNA exonuclease activity / double-stranded DNA endonuclease activity / DNA exonuclease activity / 5'-flap endonuclease activity / viral DNA genome replication / 5'-3' exonuclease activity / 5'-3' DNA exonuclease activity / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA binding / metal ion binding
Similarity search - Function
Flap endonuclease D15-like / Flap endonuclease / 5'-nuclease / DNA polymerase I-like, H3TH domain / 5'-3' exonuclease, C-terminal SAM fold / 5'-3' exonuclease, alpha-helical arch, N-terminal / 5'-3' exonuclease, N-terminal resolvase-like domain / 5'-3' exonuclease / 5'-3' exonuclease / Helix-hairpin-helix motif, class 2 ...Flap endonuclease D15-like / Flap endonuclease / 5'-nuclease / DNA polymerase I-like, H3TH domain / 5'-3' exonuclease, C-terminal SAM fold / 5'-3' exonuclease, alpha-helical arch, N-terminal / 5'-3' exonuclease, N-terminal resolvase-like domain / 5'-3' exonuclease / 5'-3' exonuclease / Helix-hairpin-helix motif, class 2 / Helix-hairpin-helix class 2 (Pol1 family) motifs / 5'-3' exonuclease, C-terminal domain superfamily / PIN-like domain superfamily / 5' to 3' exonuclease, C-terminal subdomain / DNA polymerase; domain 1 / Rossmann fold / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
: / DNA / DNA (> 10) / Flap endonuclease
Similarity search - Component
Biological speciesEscherichia phage T5 (virus)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.22 Å
AuthorsAlmalki, F.A. / Feng, M. / Zhang, J. / Sedelnikova, S.E. / Rafferty, J.B. / Sayers, J.R. / Artymiuk, P.J.
CitationJournal: Nat.Struct.Mol.Biol. / Year: 2016
Title: Direct observation of DNA threading in flap endonuclease complexes.
Authors: AlMalki, F.A. / Flemming, C.S. / Zhang, J. / Feng, M. / Sedelnikova, S.E. / Ceska, T. / Rafferty, J.B. / Sayers, J.R. / Artymiuk, P.J.
History
DepositionJan 18, 2016Deposition site: RCSB / Processing site: PDBE
Revision 1.0Jun 1, 2016Provider: repository / Type: Initial release
Revision 1.1Jun 8, 2016Group: Database references
Revision 1.2Jun 22, 2016Group: Database references
Revision 1.3Jul 20, 2016Group: Database references
Revision 1.4Jan 10, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr2_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
X: DNA (5'-D(*AP*AP*AP*AP*GP*CP*GP*TP*AP*CP*GP*C)-3')
Y: DNA (5'-D(*AP*AP*AP*AP*GP*CP*GP*TP*AP*CP*GP*C)-3')
A: Exodeoxyribonuclease
B: Exodeoxyribonuclease
hetero molecules


Theoretical massNumber of molelcules
Total (without water)70,0928
Polymers69,9124
Non-polymers1804
Water4,035224
1
X: DNA (5'-D(*AP*AP*AP*AP*GP*CP*GP*TP*AP*CP*GP*C)-3')
Y: DNA (5'-D(*AP*AP*AP*AP*GP*CP*GP*TP*AP*CP*GP*C)-3')
B: Exodeoxyribonuclease
hetero molecules


Theoretical massNumber of molelcules
Total (without water)38,8167
Polymers38,6363
Non-polymers1804
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3930 Å2
ΔGint-37 kcal/mol
Surface area16690 Å2
MethodPISA
2
X: DNA (5'-D(*AP*AP*AP*AP*GP*CP*GP*TP*AP*CP*GP*C)-3')
Y: DNA (5'-D(*AP*AP*AP*AP*GP*CP*GP*TP*AP*CP*GP*C)-3')
A: Exodeoxyribonuclease
hetero molecules


Theoretical massNumber of molelcules
Total (without water)38,7246
Polymers38,6363
Non-polymers883
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2700 Å2
ΔGint-22 kcal/mol
Surface area17480 Å2
MethodPISA
Unit cell
Length a, b, c (Å)44.720, 109.940, 127.340
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

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DNA chain / Protein , 2 types, 4 molecules XYAB

#1: DNA chain DNA (5'-D(*AP*AP*AP*AP*GP*CP*GP*TP*AP*CP*GP*C)-3')


Mass: 3680.432 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: Protein Exodeoxyribonuclease / 5'-exonuclease / T5FEN / D15 exonuclease


Mass: 31275.475 Da / Num. of mol.: 2 / Fragment: UNP residues 20-291
Source method: isolated from a genetically manipulated source
Details: Corresponds to residues 20-291 of Uniprot P06229 but with mutation D153K
Source: (gene. exp.) Escherichia phage T5 (virus) / Gene: D15 / Plasmid: pJONEX4
Details (production host): pUC19 derivative carrying a lambda promoter.
Production host: Escherichia coli (E. coli) / Strain (production host): M72
References: UniProt: P06229, exodeoxyribonuclease (lambda-induced)

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Non-polymers , 4 types, 228 molecules

#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#4: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: K
#5: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H8O3
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 224 / 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: 2.24 Å3/Da / Density % sol: 45.06 %
Crystal growTemperature: 290 K / Method: vapor diffusion, hanging drop / pH: 5.5
Details: The concentration of both oligonucleotides was adjusted to 1.1 mM for the duplex molecule by dissolving each one in 10 mM MES pH 6.5 and 50 mM KCl. They were annealed by heating to 367K for ...Details: The concentration of both oligonucleotides was adjusted to 1.1 mM for the duplex molecule by dissolving each one in 10 mM MES pH 6.5 and 50 mM KCl. They were annealed by heating to 367K for 10 minutes and allowed to cool to room temperature. Crystals with oligonucleotide 5ov4 were grown at 290K with the D153K variant of T5Fen. The resulting T5FenD153K:5ov4 structure was determined from crystals grown in 0.2 M MgCl2, 0.1 M Bis-Tris buffer pH 5.5, 25% w/v PEG 3350.

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I24 / Wavelength: 0.977 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Apr 16, 2011
Details: Please check which detector was in place at that date
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.977 Å / Relative weight: 1
ReflectionResolution: 2.22→44.72 Å / Num. all: 31727 / Num. obs: 31727 / % possible obs: 99.4 % / Redundancy: 5.7 % / Rpim(I) all: 0.038 / Rrim(I) all: 0.094 / Rsym value: 0.079 / Net I/av σ(I): 7.049 / Net I/σ(I): 13.4 / Num. measured all: 182387
Reflection shell

Diffraction-ID: 1 / Rejects: _

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured allNum. unique allRpim(I) allRsym valueNet I/σ(I) obs% possible all
2.22-2.283.10.6540.9672621770.4050.6542.294.8
2.28-2.343.70.5241.2819522380.3120.5242.998.2
2.34-2.414.40.4521.4958021890.2490.4523.799.9
2.41-2.485.50.3981.61186821390.1980.3984.999.8
2.48-2.566.40.3321.91303620520.1530.3326.199.9
2.56-2.656.70.2742.31354720230.1240.2747.5100
2.65-2.756.60.2432.61283519470.1110.2438.6100
2.75-2.876.40.1943.31180818540.0910.19410.199.9
2.87-2.996.40.15941168918350.0750.15911.999.9
2.99-3.146.60.1185.61126417010.0540.11814.9100
3.14-3.316.50.097.11061416420.0440.0918.199.9
3.31-3.516.20.0748.7965315660.0360.07420.6100
3.51-3.756.20.0669.9923614820.0310.06623.299.9
3.75-4.056.50.06110.7901613820.0280.06125.699.9
4.05-4.446.30.05511.5796412710.0260.05527.1100
4.44-4.966.10.05211.5719911730.0250.05228.1100
4.96-5.736.50.04813.4662810270.0220.04828.3100
5.73-7.025.90.0491352648900.0230.04926.699.8
7.02-9.935.80.0413.941617130.020.0429.9100
9.93-44.724.90.03515.721044260.0190.03528.698.5

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Processing

Software
NameVersionClassification
SCALA3.3.16data scaling
REFMAC5.8.0135refinement
PDB_EXTRACT3.15data extraction
xia2data reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1EXN

1exn


Resolution: 2.22→42.19 Å / Cor.coef. Fo:Fc: 0.959 / Cor.coef. Fo:Fc free: 0.932 / WRfactor Rfree: 0.2347 / WRfactor Rwork: 0.1834 / FOM work R set: 0.8335 / SU B: 14.251 / SU ML: 0.175 / SU R Cruickshank DPI: 0.3029 / SU Rfree: 0.2192 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.303 / ESU R Free: 0.219 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.2344 1595 5 %RANDOM
Rwork0.1826 ---
obs0.1851 30073 99.35 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 154.83 Å2 / Biso mean: 48.335 Å2 / Biso min: 21.96 Å2
Baniso -1Baniso -2Baniso -3
1--0.17 Å20 Å20 Å2
2---0.46 Å20 Å2
3---0.63 Å2
Refinement stepCycle: final / Resolution: 2.22→42.19 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4407 490 9 224 5130
Biso mean--65.84 46.85 -
Num. residues----568
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0070.0185050
X-RAY DIFFRACTIONr_bond_other_d0.0010.024573
X-RAY DIFFRACTIONr_angle_refined_deg1.0661.8596913
X-RAY DIFFRACTIONr_angle_other_deg0.947310527
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.3055542
X-RAY DIFFRACTIONr_dihedral_angle_2_deg30.87324.211228
X-RAY DIFFRACTIONr_dihedral_angle_3_deg11.92515813
X-RAY DIFFRACTIONr_dihedral_angle_4_deg11.0841530
X-RAY DIFFRACTIONr_chiral_restr0.0650.2742
X-RAY DIFFRACTIONr_gen_planes_refined0.0040.025358
X-RAY DIFFRACTIONr_gen_planes_other0.0010.021192
X-RAY DIFFRACTIONr_mcbond_it2.6593.0612174
X-RAY DIFFRACTIONr_mcbond_other2.6593.062173
X-RAY DIFFRACTIONr_mcangle_it3.9974.5812714
LS refinement shellResolution: 2.22→2.278 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.274 117 -
Rwork0.264 2045 -
all-2162 -
obs--94.41 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
13.764-0.6541.62120.7861-0.16663.72490.0126-0.182-0.42940.10440.16460.04820.2536-0.7765-0.17720.2067-0.0295-0.02510.2202-0.02510.162711.708512.842232.2433
20.3145-0.5901-0.50871.33561.75113.6330.0261-0.0439-0.14190.02920.0240.17630.2294-0.1989-0.05010.1421-0.0235-0.03670.1491-0.01290.140714.397616.33144.5373
30.908-0.33310.21512.57380.81131.8822-0.05640.05840.0985-0.3430.0197-0.1226-0.3270.0880.03670.1079-0.00320.00840.01730.00490.023617.559410.064810.26
40.48330.15280.04841.1710.10331.01010.1337-0.0145-0.0743-0.0611-0.1206-0.02080.0099-0.0552-0.01320.04620.0014-0.02160.13040.02140.110619.795829.366845.3881
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1X1 - 12
2X-RAY DIFFRACTION2Y1 - 12
3X-RAY DIFFRACTION3A20 - 291
4X-RAY DIFFRACTION4B20 - 291

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