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- PDB-1nk0: ADENINE-GUANINE MISMATCH AT THE POLYMERASE ACTIVE SITE -

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Basic information

Entry
Database: PDB / ID: 1nk0
TitleADENINE-GUANINE MISMATCH AT THE POLYMERASE ACTIVE SITE
Components
  • DNA POLYMERASE I
  • DNA PRIMER STRAND
  • DNA TEMPLATE STRAND
KeywordsTRANSFERASE/DNA / DNA POLYMERASE I / DNA REPLICATION / KLENOW FRAGMENT / PROTEIN-DNA COMPLEX / DNA MISMATCH / TRANSFERASE-DNA COMPLEX
Function / homology
Function and homology information


double-strand break repair via alternative nonhomologous end joining / 3'-5' exonuclease activity / DNA-templated DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA binding
Similarity search - Function
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 ...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 / 3'-5' exonuclease / 3'-5' exonuclease domain / 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 / 5' to 3' exonuclease, C-terminal subdomain / Ribonuclease H-like superfamily/Ribonuclease H / DNA polymerase; domain 1 / Nucleotidyltransferase; domain 5 / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Alpha-Beta Plaits / DNA/RNA polymerase superfamily / Up-down Bundle / 2-Layer Sandwich / Orthogonal Bundle / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
sucrose / DNA / DNA (> 10) / DNA polymerase I
Similarity search - Component
Biological speciesGeobacillus stearothermophilus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.7 Å
AuthorsJohnson, S.J. / Beese, L.S.
Citation
Journal: Cell(Cambridge,Mass.) / Year: 2004
Title: Structures of mismatch replication errors observed in a DNA polymerase.
Authors: Johnson, S.J. / Beese, L.S.
#1: Journal: Proc.Natl.Acad.Sci.USA / Year: 2003
Title: Processive DNA synthesis observed in a polymerase crystal suggests a mechanism for the prevention of frameshift mutations.
Authors: Johnson, S.J. / Taylor, J.S. / Beese, L.S.
#2: Journal: Nature / Year: 1998
Title: Visualizing DNA Replication in a Catalytically Active Bacillus DNA Polymerase Crystal
Authors: Kiefer, J.R. / Mao, C. / Braman, J.C. / Beese, L.S.
#3: Journal: Structure / Year: 1997
Title: Crystal Structure of a Thermostable Bacillus DNA Polymerase I Large Fragment at 2.1 A Resolution
Authors: Kiefer, J.R. / Mao, C. / Hansen, C.J. / Basehore, S.L. / Hogrefe, H.H. / Braman, J.C. / Beese, L.S.
History
DepositionJan 2, 2003Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 30, 2004Provider: repository / Type: Initial release
Revision 1.1Apr 29, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 2.0Jul 29, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Non-polymer description / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / entity / entity_name_com / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_molecule_features / pdbx_nonpoly_scheme / struct_asym / struct_conn / struct_conn_type / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.type_symbol / _chem_comp.formula / _chem_comp.formula_weight / _chem_comp.id / _chem_comp.mon_nstd_flag / _chem_comp.name / _chem_comp.type / _entity.formula_weight / _entity.pdbx_description / _entity.pdbx_number_of_molecules / _entity.type / _struct_asym.entity_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 2.1Aug 16, 2023Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: DNA PRIMER STRAND
C: DNA TEMPLATE STRAND
A: DNA POLYMERASE I
hetero molecules


Theoretical massNumber of molelcules
Total (without water)75,0639
Polymers74,1413
Non-polymers9226
Water11,854658
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)88.124, 93.609, 104.746
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121
DetailsExists as a monomer. One molecule per asymmetric unit.

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Components

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DNA chain , 2 types, 2 molecules BC

#1: DNA chain DNA PRIMER STRAND


Mass: 3367.225 Da / Num. of mol.: 1 / Source method: obtained synthetically
#2: DNA chain DNA TEMPLATE STRAND


Mass: 4600.983 Da / Num. of mol.: 1 / Source method: obtained synthetically

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Protein / Sugars , 2 types, 2 molecules A

#3: Protein DNA POLYMERASE I / EC 2.7.7.7


Mass: 66172.844 Da / Num. of mol.: 1
Fragment: BACILLUS FRAGMENT (ANALOGOUS TO THE E. COLI KLENOW FRAGMENT)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacillus stearothermophilus (bacteria)
Plasmid: PET-30A(+) / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)PLYSS / References: UniProt: P52026, DNA-directed DNA polymerase
#4: Polysaccharide beta-D-fructofuranose-(2-1)-alpha-D-glucopyranose / sucrose


Type: oligosaccharide, Oligosaccharide / Class: Nutrient / Mass: 342.297 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: oligosaccharide with reducing-end-to-reducing-end glycosidic bond
References: sucrose
DescriptorTypeProgram
DFrufb2-1DGlcpaGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,2,1/[ha122h-2b_2-5][a2122h-1a_1-5]/1-2/a2-b1WURCSPDB2Glycan 1.1.0
[][b-D-Fruf]{[(2+1)][a-D-Glcp]{}}LINUCSPDB-CARE

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Non-polymers , 3 types, 663 molecules

#5: Chemical ChemComp-MES / 2-(N-MORPHOLINO)-ETHANESULFONIC ACID


Mass: 195.237 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H13NO4S / Comment: pH buffer*YM
#6: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: SO4
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 658 / Source method: isolated from a natural source / Formula: H2O

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Details

Compound detailsTHE POLYMERASE-DNA COMPLEX ADOPTS A DISTORTED OPEN CONFORMATION WITH AN ADENINE-GUANINE MISMATCH ...THE POLYMERASE-DNA COMPLEX ADOPTS A DISTORTED OPEN CONFORMATION WITH AN ADENINE-GUANINE MISMATCH BOUND AT THE POLYMERASE POST-INSERTION SITE. THE MISMATCH ADOPTS AN ANTI-ANTI CONFORMATION.

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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: 2.65 Å3/Da / Density % sol: 53.17 %
Crystal growTemperature: 290 K / Method: vapor diffusion, hanging drop / pH: 5.8
Details: AMMONIUM SULFATE, MAGNESIUM SULFATE, MPD, MES, pH 5.80, VAPOR DIFFUSION, HANGING DROP, temperature 290K
Components of the solutions
IDNameCrystal-IDSol-ID
1AMMONIUM SULFAT11
2MAGNESIUM SULFATE11
3MPD11
4MES11
5water11
6AMMONIUM SULFATE12
7MAGNESIUM SULFATE12
8MPD12
Crystal grow
*PLUS
pH: 5.8 / Method: vapor diffusion, hanging drop
Details: Johnson, S.J., (2003) Proc.Natl.Acad.Sci.USA, 100, 3895.
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDDetailsChemical formula
112 mg/mlprotein1drop
249 %satammonium sulfate1reservoir
32.5 %(v/v)1reservoir
4100 mM1reservoirpH5.8
510 mM1reservoirMgSO4
61
71
81

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Data collection

Diffraction
IDMean temperature (K)Crystal-ID
11001
21
Diffraction sourceSource: SYNCHROTRON / Site: NSLS / Beamline: X12B / Wavelength: 1.0001 Å
DetectorType: ADSC QUANTUM 4 / Detector: CCD / Date: Oct 13, 2000
RadiationMonochromator: Si CRYSTALS / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.0001 Å / Relative weight: 1
ReflectionResolution: 1.7→50 Å / Num. all: 94629 / Num. obs: 94629 / % possible obs: 98.2 % / Observed criterion σ(F): -3 / Observed criterion σ(I): -3 / Redundancy: 3.1 % / Biso Wilson estimate: 15.3 Å2 / Rsym value: 0.047 / Net I/σ(I): 20.2
Reflection shellResolution: 1.7→1.76 Å / Redundancy: 2.5 % / Mean I/σ(I) obs: 3.3 / Num. unique all: 8835 / Rsym value: 0.26 / % possible all: 92.9
Reflection
*PLUS
Num. measured all: 292563 / Rmerge(I) obs: 0.047
Reflection shell
*PLUS
Highest resolution: 1.7 Å / Rmerge(I) obs: 0.26

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Processing

Software
NameClassification
DENZOdata reduction
SCALEPACKdata scaling
CNSrefinement
CNSphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 2BDP

2bdp


Resolution: 1.7→28.32 Å / Rfactor Rfree error: 0.003 / Data cutoff high absF: 2153708.32 / Data cutoff high rms absF: 2153708.32 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 2 / Stereochemistry target values: Engh & Huber
Details: THE BACILLUS POLYMERASE WAS CO- CRYSTALLIZED WITH A DNA CONTAINING AN 11 BASE PAIR DUPLEX AND A 4 BASE 5' TEMPLATE OVERHANG. THE LAST THREE BASES IN THE OVERHANG ARE DISORDERED AND ARE NOT ...Details: THE BACILLUS POLYMERASE WAS CO- CRYSTALLIZED WITH A DNA CONTAINING AN 11 BASE PAIR DUPLEX AND A 4 BASE 5' TEMPLATE OVERHANG. THE LAST THREE BASES IN THE OVERHANG ARE DISORDERED AND ARE NOT INCLUDED IN THE MODEL. AN ADENINE-GUANINE MISMATCH IS LOCATED AT THE 3' PRIMER TERMINUS OF THE DNA DUPLEX. ELECTRON DENSITY WAS OBSERVED FOR ALL PROTEIN SIDE CHAINS EXCEPT LYSINE 298, WHICH WAS MODELLED TO THE BETA CARBON.
RfactorNum. reflection% reflectionSelection details
Rfree0.209 4513 5.1 %RANDOM
Rwork0.189 ---
all0.203 91904 --
obs0.203 88865 92.8 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 44.3887 Å2 / ksol: 0.378445 e/Å3
Displacement parametersBiso mean: 20.8 Å2
Baniso -1Baniso -2Baniso -3
1-6.25 Å20 Å20 Å2
2---3.15 Å20 Å2
3----3.1 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.2 Å0.18 Å
Luzzati d res low-5 Å
Luzzati sigma a0.14 Å0.12 Å
Refinement stepCycle: LAST / Resolution: 1.7→28.32 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4650 466 55 658 5829
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.008
X-RAY DIFFRACTIONc_bond_d_na
X-RAY DIFFRACTIONc_bond_d_prot
X-RAY DIFFRACTIONc_angle_d
X-RAY DIFFRACTIONc_angle_d_na
X-RAY DIFFRACTIONc_angle_d_prot
X-RAY DIFFRACTIONc_angle_deg1.2
X-RAY DIFFRACTIONc_angle_deg_na
X-RAY DIFFRACTIONc_angle_deg_prot
X-RAY DIFFRACTIONc_dihedral_angle_d22.3
X-RAY DIFFRACTIONc_dihedral_angle_d_na
X-RAY DIFFRACTIONc_dihedral_angle_d_prot
X-RAY DIFFRACTIONc_improper_angle_d0.9
X-RAY DIFFRACTIONc_improper_angle_d_na
X-RAY DIFFRACTIONc_improper_angle_d_prot
X-RAY DIFFRACTIONc_mcbond_it0.691.5
X-RAY DIFFRACTIONc_mcangle_it1.132
X-RAY DIFFRACTIONc_scbond_it1.292
X-RAY DIFFRACTIONc_scangle_it2.032.5
LS refinement shellResolution: 1.7→1.76 Å / Rfactor Rfree error: 0.012 / Total num. of bins used: 10
RfactorNum. reflection% reflection
Rfree0.23 399 5.5 %
Rwork0.223 6914 -
obs--77.1 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PROTEIN_REP.PARAMPROTEIN.TOP
X-RAY DIFFRACTION2DNA-RNA-MULTI-ENDO.PARAMDNA-RNA-MULTI-ENDO.TOP
X-RAY DIFFRACTION3WATER_REP.PARAMWATER.TOP
X-RAY DIFFRACTION4ION.PARAMION.TOP
X-RAY DIFFRACTION5SUCROSE-MES.PARAMSUCROSE-MES.TOP
Refinement
*PLUS
Highest resolution: 1.7 Å / Lowest resolution: 50 Å / Rfactor Rfree: 0.213 / Rfactor Rwork: 0.193 / % reflection Rfree: 5 %
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_dihedral_angle_d
X-RAY DIFFRACTIONc_dihedral_angle_deg22.3
X-RAY DIFFRACTIONc_improper_angle_d
X-RAY DIFFRACTIONc_improper_angle_deg0.9
LS refinement shell
*PLUS
Rfactor Rfree: 0.247 / Rfactor Rwork: 0.246

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