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- PDB-5w6k: Structure of mutant Taq Polymerase incorporating unnatural base p... -

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Basic information

Entry
Database: PDB / ID: 5w6k
TitleStructure of mutant Taq Polymerase incorporating unnatural base pairs Z:P
Components
  • DNA (5'-D(*GP*AP*CP*CP*AP*CP*GP*GP*CP*GP*CP*(DOC))-3')
  • DNA (5'-D(P*(1WA)P*GP*GP*CP*GP*CP*CP*GP*TP*GP*GP*TP*C)-3')
  • DNA polymerase I, thermostable
KeywordsDNA binding protein/DNA / Mutant Taq Polymerase / Unnatural base pairs / AEGIS / ternary complex / Klentaq / Binary complex / TRANSFERASE-DNA complex / DNA binding protein-DNA complex
Function / homology
Function and homology information


nucleoside binding / hydrolase activity, acting on ester bonds / double-strand break repair via alternative nonhomologous end joining / DNA-templated DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA binding
Similarity search - Function
Taq polymerase, thermostable, exonuclease region / Taq polymerase, exonuclease / 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 / Taq DNA Polymerase; Chain T, domain 4 / Taq DNA Polymerase; Chain T, domain 4 ...Taq polymerase, thermostable, exonuclease region / Taq polymerase, exonuclease / 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 / Taq DNA Polymerase; Chain T, domain 4 / Taq DNA Polymerase; Chain T, domain 4 / DNA polymerase 1 / Alpha-Beta Plaits - #370 / Helix-hairpin-helix motif, class 2 / Helix-hairpin-helix class 2 (Pol1 family) motifs / 5'-3' exonuclease, C-terminal domain superfamily / DNA polymerase A / DNA polymerase family A / DNA-directed DNA polymerase, family A, conserved site / DNA polymerase family A signature. / DNA-directed DNA polymerase, family A, palm domain / DNA polymerase A domain / PIN-like domain superfamily / Helix-hairpin-helix DNA-binding motif, class 1 / Helix-hairpin-helix DNA-binding motif class 1 / Ribonuclease H-like superfamily/Ribonuclease H / Nucleotidyltransferase; domain 5 / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Alpha-Beta Plaits / DNA/RNA polymerase superfamily / Up-down Bundle / 2-Layer Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
Chem-A5J / DNA / DNA (> 10) / DNA polymerase I, thermostable
Similarity search - Component
Biological speciesThermus aquaticus (bacteria)
Escherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.339 Å
AuthorsSingh, I. / Georgiadis, M.M.
CitationJournal: Nucleic Acids Res. / Year: 2018
Title: Snapshots of an evolved DNA polymerase pre- and post-incorporation of an unnatural nucleotide.
Authors: Singh, I. / Laos, R. / Hoshika, S. / Benner, S.A. / Georgiadis, M.M.
History
DepositionJun 16, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 18, 2018Provider: repository / Type: Initial release
Revision 1.1Sep 19, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Oct 4, 2023Group: 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 / struct_conn_type
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.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_conn_type.id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA polymerase I, thermostable
B: DNA (5'-D(*GP*AP*CP*CP*AP*CP*GP*GP*CP*GP*CP*(DOC))-3')
C: DNA (5'-D(P*(1WA)P*GP*GP*CP*GP*CP*CP*GP*TP*GP*GP*TP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)69,0696
Polymers68,5073
Non-polymers5623
Water3,009167
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5360 Å2
ΔGint-20 kcal/mol
Surface area24860 Å2
MethodPISA
Unit cell
Length a, b, c (Å)109.306, 109.306, 90.871
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number152
Space group name H-MP3121
Components on special symmetry positions
IDModelComponents
11A-1133-

HOH

21A-1137-

HOH

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Components

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Protein , 1 types, 1 molecules A

#1: Protein DNA polymerase I, thermostable / Taq polymerase 1


Mass: 60862.902 Da / Num. of mol.: 1 / Fragment: UNP residues 293-831
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus aquaticus (bacteria) / Gene: polA, pol1 / Production host: Escherichia coli (E. coli) / References: UniProt: P19821, DNA-directed DNA polymerase

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DNA chain , 2 types, 2 molecules BC

#2: DNA chain DNA (5'-D(*GP*AP*CP*CP*AP*CP*GP*GP*CP*GP*CP*(DOC))-3')


Mass: 3617.371 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#3: DNA chain DNA (5'-D(P*(1WA)P*GP*GP*CP*GP*CP*CP*GP*TP*GP*GP*TP*C)-3')


Mass: 4026.612 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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Non-polymers , 3 types, 170 molecules

#4: Chemical ChemComp-A5J / (1R)-1-[6-amino-5-(dihydroxyamino)-2-hydroxypyridin-3-yl]-1,4-anhydro-2-deoxy-5-O-[(S)-hydroxy{[(S)-hydroxy(phosphonooxy)phosphoryl]oxy}phosphoryl]-D-erythro-pentitol


Mass: 513.182 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H18N3O15P3 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 167 / 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: 2.36 Å3/Da / Density % sol: 47.94 %
Crystal growTemperature: 277.16 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: 19 % PEG 4000, 0.2 M ammonium acetate, 0.01 M magnesium acetate, 0.05 M sodium cacodylate pH 6.5

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 19-ID / Wavelength: 0.98 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Mar 2, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.98 Å / Relative weight: 1
ReflectionResolution: 2.339→41.978 Å / Num. obs: 26672 / % possible obs: 99.3 % / Redundancy: 6.1 % / Biso Wilson estimate: 29.27 Å2 / Rmerge(I) obs: 0.112 / Rpim(I) all: 0.046 / Rrim(I) all: 0.122 / Χ2: 0.962 / Net I/σ(I): 12.2
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
2.35-2.393.50.39912870.8220.2280.4630.88396.5
2.39-2.4340.39912860.860.2140.4550.8598.1
2.43-2.484.60.38513080.870.1940.4330.8899.5
2.48-2.535.10.34513220.9160.1640.3840.88699.5
2.53-2.595.30.31913110.9270.1480.3530.92499.5
2.59-2.655.20.29713200.9390.1390.3290.82499.5
2.65-2.716.60.28513100.9510.1190.310.87299.5
2.71-2.796.80.25713300.9620.1050.2780.86199.8
2.79-2.876.90.22313330.9690.0910.2420.86299.9
2.87-2.966.70.19313380.970.080.2090.999.7
2.96-3.076.50.16213300.9780.0670.1760.89399.9
3.07-3.1960.13613220.9810.0590.1490.94499.7
3.19-3.336.50.11813530.9870.0490.1281.02599.8
3.33-3.517.30.10613240.9890.0410.1141.03799.6
3.51-3.737.20.113490.9890.040.1071.03499.7
3.73-4.0270.09613230.9850.0390.1041.00999.6
4.02-4.426.40.09313610.9880.0390.1021.0499.6
4.42-5.067.40.09513700.9880.0380.1020.9899.6
5.06-6.386.60.09813600.9860.040.1061.01599.2
6.38-1007.20.11314350.9820.0440.1211.25598.6

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
PHENIX1.11.1_2575refinement
SCALEPACKdata scaling
MOLREPphasing
PDB_EXTRACT3.22data extraction
DENZOdata collection
HKL-2000data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3RTV

3rtv


Resolution: 2.339→41.978 Å / SU ML: 0.37 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 27.26
RfactorNum. reflection% reflection
Rfree0.2746 1229 4.75 %
Rwork0.2242 --
obs0.2265 25878 96.22 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 146.93 Å2 / Biso mean: 44.7161 Å2 / Biso min: 10.11 Å2
Refinement stepCycle: final / Resolution: 2.339→41.978 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4128 510 47 167 4852
Biso mean--32.72 28.99 -
Num. residues----547
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0034819
X-RAY DIFFRACTIONf_angle_d0.596635
X-RAY DIFFRACTIONf_chiral_restr0.039734
X-RAY DIFFRACTIONf_plane_restr0.003774
X-RAY DIFFRACTIONf_dihedral_angle_d14.4482818
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 9

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.3386-2.43220.34781160.28842062217874
2.4322-2.54290.33431160.27452726284296
2.5429-2.6770.34791320.27842778291098
2.677-2.84460.32161550.270527852940100
2.8446-3.06420.30751880.250827622950100
3.0642-3.37250.31941360.239528462982100
3.3725-3.86020.25191490.207728242973100
3.8602-4.86230.19661310.178828662997100
4.8623-41.98470.2391060.19743000310699
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)
11.27410.11540.10610.828-0.02460.8239-0.0904-0.0337-0.7241-0.26080.025-0.37650.21450.07560.06270.2916-0.06240.14340.2177-0.00650.551439.3858-41.4522-18.0278
20.47890.42490.00480.4973-0.20430.38640.1679-0.08270.2585-0.0566-0.05970.0939-0.8041-0.0549-0.0710.6238-0.02540.10670.2008-0.02240.328435.9072-11.00430.8575
30.3148-0.0049-0.25580.1219-0.34381.2375-0.1654-0.41670.18390.10140.30530.2226-0.6106-0.83990.38030.5030.4331-0.04290.98170.09370.29479.8414-16.9606-7.5362
40.89460.2926-0.39580.79090.21421.4972-0.09630.0795-0.053-0.26590.21090.0684-0.3383-0.727-0.11930.28270.02450.00020.4150.08750.207820.9163-25.6287-16.6075
55.2442-1.5702-0.12980.60.36020.79790.03690.4578-0.1785-0.0537-0.1049-0.0178-0.0906-0.1026-0.03260.27820.1222-0.12950.71790.15330.761248.5504-29.91455.2064
62.28771.03830.91571.45490.62630.9206-0.0249-0.28790.01010.09470.07920.0473-0.221-0.2878-0.08970.20840.08770.03340.29480.060.190731.041-22.27270.874
71.31060.07371.53120.337-0.4192.56110.09960.2420.0854-0.1101-0.1032-0.0623-0.3175-0.21860.02690.34320.13730.03790.28380.06010.214429.0776-19.33921.3978
82.473-2.40270.67022.5089-0.83180.3677-0.1991-0.26160.05880.35620.22620.0248-0.1215-0.11660.02040.26980.1844-0.16370.4210.03210.425146.6825-27.525914.4848
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 296 through 448 )A296 - 448
2X-RAY DIFFRACTION2chain 'A' and (resid 449 through 604 )A449 - 604
3X-RAY DIFFRACTION3chain 'A' and (resid 605 through 713 )A605 - 713
4X-RAY DIFFRACTION4chain 'A' and (resid 714 through 831 )A714 - 832
5X-RAY DIFFRACTION5chain 'B' and (resid 101 through 104 )B101 - 104
6X-RAY DIFFRACTION6chain 'C' and (resid 204 through 212 )C204 - 212
7X-RAY DIFFRACTION7chain 'B' and (resid 105 through 111 )B105 - 113
8X-RAY DIFFRACTION8chain 'C' and (resid 213 through 216 )C213 - 216

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