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- PDB-5w6q: Structural basis for recognition of artificial DNA by an evolved ... -

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Basic information

Entry
Database: PDB / ID: 5w6q
TitleStructural basis for recognition of artificial DNA by an evolved KlenTaq variant
Components
  • DNA (5'-D(*GP*AP*CP*CP*AP*CP*GP*GP*CP*GP*CP*(1W5))-3')
  • DNA (5'-D(P*GP*(1WA)P*GP*CP*GP*CP*CP*GP*TP*GP*GP*TP*C)-3')
  • DNA polymerase I, thermostable
KeywordsDNA BINDING PROTEIN/DNA / Protein-DNA / AEGIS / unnatural base pair / host-guest system / transferase-dna complex / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


nucleoside binding / double-strand break repair via alternative nonhomologous end joining / hydrolase activity, acting on ester bonds / 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
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: 2.66 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 / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_leaving_atom_flag

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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*(1W5))-3')
D: DNA (5'-D(P*GP*(1WA)P*GP*CP*GP*CP*CP*GP*TP*GP*GP*TP*C)-3')
C: DNA polymerase I, thermostable
E: DNA (5'-D(*GP*AP*CP*CP*AP*CP*GP*GP*CP*GP*CP*(1W5))-3')
F: DNA (5'-D(P*GP*(1WA)P*GP*CP*GP*CP*CP*GP*TP*GP*GP*TP*C)-3')
G: DNA polymerase I, thermostable
H: DNA (5'-D(*GP*AP*CP*CP*AP*CP*GP*GP*CP*GP*CP*(1W5))-3')
I: DNA (5'-D(P*GP*(1WA)P*GP*CP*GP*CP*CP*GP*TP*GP*GP*TP*C)-3')


Theoretical massNumber of molelcules
Total (without water)205,7019
Polymers205,7019
Non-polymers00
Water5,314295
1
A: DNA polymerase I, thermostable
B: DNA (5'-D(*GP*AP*CP*CP*AP*CP*GP*GP*CP*GP*CP*(1W5))-3')
D: DNA (5'-D(P*GP*(1WA)P*GP*CP*GP*CP*CP*GP*TP*GP*GP*TP*C)-3')


Theoretical massNumber of molelcules
Total (without water)68,5673
Polymers68,5673
Non-polymers00
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4730 Å2
ΔGint-17 kcal/mol
Surface area24830 Å2
MethodPISA
2
C: DNA polymerase I, thermostable
E: DNA (5'-D(*GP*AP*CP*CP*AP*CP*GP*GP*CP*GP*CP*(1W5))-3')
F: DNA (5'-D(P*GP*(1WA)P*GP*CP*GP*CP*CP*GP*TP*GP*GP*TP*C)-3')


Theoretical massNumber of molelcules
Total (without water)68,5673
Polymers68,5673
Non-polymers00
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4770 Å2
ΔGint-19 kcal/mol
Surface area24890 Å2
MethodPISA
3
G: DNA polymerase I, thermostable
H: DNA (5'-D(*GP*AP*CP*CP*AP*CP*GP*GP*CP*GP*CP*(1W5))-3')
I: DNA (5'-D(P*GP*(1WA)P*GP*CP*GP*CP*CP*GP*TP*GP*GP*TP*C)-3')


Theoretical massNumber of molelcules
Total (without water)68,5673
Polymers68,5673
Non-polymers00
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4650 Å2
ΔGint-18 kcal/mol
Surface area25100 Å2
MethodPISA
Unit cell
Length a, b, c (Å)199.282, 114.611, 90.516
Angle α, β, γ (deg.)90.000, 91.880, 90.000
Int Tables number5
Space group name H-MC121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11chain D and segid D
21chain F and segid F
31chain I and segid I

NCS domain segments:
Dom-IDComponent-IDEns-IDSelection detailsAuth asym-IDAuth seq-ID
111chain D and segid DD0
211chain F and segid FF0
311chain I and segid II0

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Components

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


Mass: 60862.902 Da / Num. of mol.: 3
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
#2: DNA chain DNA (5'-D(*GP*AP*CP*CP*AP*CP*GP*GP*CP*GP*CP*(1W5))-3')


Mass: 3677.380 Da / Num. of mol.: 3 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#3: DNA chain DNA (5'-D(P*GP*(1WA)P*GP*CP*GP*CP*CP*GP*TP*GP*GP*TP*C)-3')


Mass: 4026.612 Da / Num. of mol.: 3 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 295 / 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: 2.51 Å3/Da / Density % sol: 51.02 %
Crystal growTemperature: 277.15 K / Method: vapor diffusion, hanging drop / pH: 6 / Details: 14 % PEG 8000, 0.2 M Mg formate, 0.1 M MES pH 6.0

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 31-ID / Wavelength: 0.9793 Å
DetectorType: RAYONIX MX-225 / Detector: CCD / Date: Feb 24, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9793 Å / Relative weight: 1
ReflectionResolution: 2.662→49.132 Å / Num. all: 57514 / Num. obs: 57623 / % possible obs: 98.8 % / Redundancy: 3.4 % / Biso Wilson estimate: 39.82 Å2 / Rpim(I) all: 0.063 / Rrim(I) all: 0.118 / Rsym value: 0.082 / Net I/av σ(I): 8.9 / Net I/σ(I): 11.8 / Num. measured all: 194839
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured allNum. unique allRpim(I) allRrim(I) allRsym valueNet I/σ(I) obs% possible all
2.66-2.812.70.4521.72212182980.3490.5990.4522.297.8
2.81-2.9830.3082.42357179050.2350.4180.3083.398.8
2.98-3.183.30.23.72447874620.1470.2710.25.299.2
3.18-3.443.50.12562465169710.0890.1690.1258.599.4
3.44-3.763.70.098.32365864230.0620.120.0912.699.1
3.76-4.213.80.06112.22178457880.0420.0810.06116.999.1
4.21-4.863.70.04715.61900550980.0320.0630.04721.998.5
4.86-5.953.70.04515.21626843530.0310.0610.04522.399
5.95-8.423.70.03420.41261033700.0240.0460.03425.799.1
8.42-99.3393.60.02129.2669318460.0150.0280.02135.496.8

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
PHENIX1.9_1692refinement
SCALAdata scaling
XDSdata collection
PDB_EXTRACT3.22data extraction
MOSFLMdata reduction
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3SZ2

3sz2


Resolution: 2.66→49.132 Å / SU ML: 0.36 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 27.29 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2471 2818 4.89 %
Rwork0.2113 54805 -
obs0.2131 57623 98.53 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 89.05 Å2 / Biso mean: 39.9312 Å2 / Biso min: 17.79 Å2
Refinement stepCycle: final / Resolution: 2.66→49.132 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms12321 1542 0 295 14158
Biso mean---35.03 -
Num. residues----1638
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00214329
X-RAY DIFFRACTIONf_angle_d0.70319733
X-RAY DIFFRACTIONf_chiral_restr0.0492193
X-RAY DIFFRACTIONf_plane_restr0.0032308
X-RAY DIFFRACTIONf_dihedral_angle_d15.4225486
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11D342X-RAY DIFFRACTION0.394TORSIONAL
12F342X-RAY DIFFRACTION0.394TORSIONAL
13I342X-RAY DIFFRACTION0.394TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 20

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.66-2.70590.36911440.29782663280797
2.7059-2.75510.33061210.28222749287098
2.7551-2.80810.32481470.27522710285798
2.8081-2.86540.31111310.27732747287898
2.8654-2.92770.29891230.2762734285799
2.9277-2.99580.36461280.27512702283099
2.9958-3.07070.36141220.2762782290499
3.0707-3.15370.28471130.25932790290399
3.1537-3.24650.27951470.25392753290099
3.2465-3.35120.3051660.23432728289499
3.3512-3.4710.29051260.21662772289899
3.471-3.60990.24341510.21482736288799
3.6099-3.77410.22041240.19862729285399
3.7741-3.9730.24471400.18852766290699
3.973-4.22180.20711750.17322713288899
4.2218-4.54760.19521160.16552755287198
4.5476-5.00480.22021570.17212747290498
5.0048-5.72810.22781550.17992754290999
5.7281-7.21320.21321640.19222748291299
7.2132-49.14010.18241680.18072727289596

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