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- PDB-1l3u: Crystal Structure of Bacillus DNA Polymerase I Fragment product c... -

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Basic information

Entry
Database: PDB / ID: 1l3u
TitleCrystal Structure of Bacillus DNA Polymerase I Fragment product complex with 11 base pairs of duplex DNA following addition of a dTTP and a dATP residue.
Components
  • 5'-D(*GP*AP*C*GP*TP*AP*CP*GP*TP*GP*AP*TP*CP*GP*CP*A)-3'
  • 5'-D(*GP*CP*GP*AP*TP*CP*AP*CP*GP*TP*A)-3'
  • DNA Polymerase I
KeywordsTRANSFERASE/DNA / DNA Polymerase I / DNA Replication / Klenow Fragment / Protein-DNA Complex / TRANSFERASE-DNA COMPLEX
Function / homology
Function and homology information


3'-5' exonuclease activity / DNA-templated DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA repair / 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: 1.8 Å
AuthorsJohnson, S.J. / Taylor, J.S. / Beese, L.S.
Citation
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.
#1: 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.
#2: 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
DepositionMar 1, 2002Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 25, 2003Provider: repository / Type: Initial release
Revision 1.1Apr 28, 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 / pdbx_struct_conn_angle / 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_atom_id / _atom_site.label_comp_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.type / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value
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
Remark 999SEQUENCE The DNA polymerase gene was cloned from an organism that was classified as a thermostable ...SEQUENCE The DNA polymerase gene was cloned from an organism that was classified as a thermostable strain of Bacillus stearothermophilus by ribosomal RNA sequencing. However, this particular gene has much greater homology with the analogous gene from Bacillus caldotenax. The sequence matches residues 297-877 of SwissProt entry, Q04957, whose source is Bacillus caldotenax. There are 6 discrepancies with the Q04957 entry, at residues 456, 505, 512, 550, and 823, as well as a deletion of SwissProt residue number 576.

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)75,4519
Polymers74,4543
Non-polymers9976
Water10,467581
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)86.429, 93.018, 106.137
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 5'-D(*GP*CP*GP*AP*TP*CP*AP*CP*GP*TP*A)-3'


Mass: 3358.211 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: primer strand
#2: DNA chain 5'-D(*GP*AP*C*GP*TP*AP*CP*GP*TP*GP*AP*TP*CP*GP*CP*A)-3'


Mass: 4923.204 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: template strand

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Protein / Sugars , 2 types, 3 molecules A

#3: Protein DNA Polymerase I /


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)
Description: This protein was isolated from an as yet unnamed novel strain of Bacillus stearothermophilus (see Ref 2).
Production host: Escherichia coli (E. coli) / 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.: 2
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, 585 molecules

#5: Chemical ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: SO4
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 581 / 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: 2.85 Å3/Da / Density % sol: 56.85 %
Crystal growTemperature: 290 K / Method: vapor diffusion, hanging drop / pH: 5.8
Details: Ammonium Sulfate, magnesium chloride, MPD, MES, pH 5.8, VAPOR DIFFUSION, HANGING DROP, temperature 290K
Components of the solutions
IDNameCrystal-IDSol-ID
1(NH4)2(SO4)11
2MgCl211
3MPD11
4MES11
5MgCl212
6MPD12
7MES12
Crystal grow
*PLUS
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

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: NSLS / Beamline: X12B / Wavelength: 1.000034 Å
DetectorType: ADSC QUANTUM 4 / Detector: CCD / Date: Mar 18, 2000
RadiationMonochromator: Si Crystals / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.000034 Å / Relative weight: 1
ReflectionResolution: 1.8→50 Å / Num. all: 79466 / Num. obs: 72778 / % possible obs: 90.3 % / Observed criterion σ(I): -3 / Redundancy: 3.4 % / Biso Wilson estimate: 11.7 Å2 / Rmerge(I) obs: 0.073 / Net I/σ(I): 10.8
Reflection shellResolution: 1.8→1.86 Å / Redundancy: 4.1 % / Rmerge(I) obs: 0.362 / Mean I/σ(I) obs: 1.6 / Num. unique all: 7722 / % possible all: 96.5
Reflection
*PLUS
Highest resolution: 1.8 Å / Lowest resolution: 50 Å / Num. obs: 79466 / % possible obs: 79.4 % / Num. measured all: 358326
Reflection shell
*PLUS
% possible obs: 48.4 %

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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: 1.8→46.09 Å / Rfactor Rfree error: 0.004 / Data cutoff high absF: 2246054.7 / 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 a 9 base pair duplex and a 6 base 5' template overhang. The crystal was subsequently placed into a stabilizing solution ...Details: The Bacillus polymerase was co-crystallized with a DNA containing a 9 base pair duplex and a 6 base 5' template overhang. The crystal was subsequently placed into a stabilizing solution containing dTTP and dATP. The resulting product complex contained 11 base pairs of duplex DNA and a 4 base 5' template overhang. The last three bases in the overhang are disordered and are not included in the model. A single base 3' overhang on the template strand (chain C) assured that the DNA duplex was not bound backwards by the polymerase during crystallization. Electron density was observed for all protein side chains except lysine 4298, which was modelled to the beta carbon. The magnesium at position 920 was assigned due to the low refined b-factor and comparison with a related Klentaq polymerase structure (3KTQ). However, the resolution of the structure prevents a definitive assignment between water and magnesium.
RfactorNum. reflection% reflectionSelection details
Rfree0.2281 3234 5.1 %RANDOM
Rwork0.1942 ---
obs0.1959 63346 79.4 %-
all-71448 --
Solvent computationSolvent model: FLAT MODEL / Bsol: 41.07 Å2 / ksol: 0.38 e/Å3
Displacement parametersBiso mean: 23.9 Å2
Baniso -1Baniso -2Baniso -3
1-6.43 Å20 Å20 Å2
2---5.26 Å20 Å2
3----1.17 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.24 Å0.2 Å
Luzzati d res low-5 Å
Luzzati sigma a0.28 Å0.24 Å
Refinement stepCycle: LAST / Resolution: 1.8→46.09 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4650 488 62 581 5781
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.005
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_d22.5
X-RAY DIFFRACTIONc_dihedral_angle_d_na
X-RAY DIFFRACTIONc_dihedral_angle_d_prot
X-RAY DIFFRACTIONc_improper_angle_d0.87
X-RAY DIFFRACTIONc_improper_angle_d_na
X-RAY DIFFRACTIONc_improper_angle_d_prot
X-RAY DIFFRACTIONc_mcbond_it0.861.5
X-RAY DIFFRACTIONc_mcangle_it1.172
X-RAY DIFFRACTIONc_scbond_it1.032
X-RAY DIFFRACTIONc_scangle_it1.582.5
LS refinement shellResolution: 1.8→1.91 Å / Rfactor Rfree error: 0.016 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.311 374 5.3 %
Rwork0.283 6660 -
obs--54 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PROTEIN_REP.PAPROTEIN.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.PARSUCROSE.TOP
Refinement
*PLUS
Lowest resolution: 50 Å / % reflection Rfree: 5 % / Rfactor Rfree: 0.228 / Rfactor Rwork: 0.194
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_dihedral_angle_d
X-RAY DIFFRACTIONc_dihedral_angle_deg22.5
X-RAY DIFFRACTIONc_improper_angle_d
X-RAY DIFFRACTIONc_improper_angle_deg0.87

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