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- PDB-1lv5: Crystal Structure of the Closed Conformation of Bacillus DNA Poly... -

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Basic information

Entry
Database: PDB / ID: 1lv5
TitleCrystal Structure of the Closed Conformation of Bacillus DNA Polymerase I Fragment Bound to DNA and dCTP
Components
  • 5'-D(*AP*CP*GP*TP*CP*GP*CP*TP*GP*AP*TP*CP*CP*G)-3'
  • 5'-D(*GP*GP*AP*TP*CP*AP*GP*CP*GP*A)-3'
  • DNA POLYMERASE I
KeywordsTRANSFERASE/DNA / DNA POLYMERASE I / DNA REPLICATION / KLENOW FRAGMENT / PROTEIN-DNA-DNTP COMPLEX / CLOSED CONFORMATION / 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
2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE / : / DNA / DNA (> 10) / DNA polymerase I
Similarity search - Component
Biological speciesGeobacillus stearothermophilus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.95 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
DepositionMay 24, 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 1.3Feb 14, 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 / struct_site
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_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 / _struct_conn.pdbx_dist_value / _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.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_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)148,22013
Polymers146,9596
Non-polymers1,2617
Water3,351186
1
C: 5'-D(*GP*GP*AP*TP*CP*AP*GP*CP*GP*A)-3'
D: 5'-D(*AP*CP*GP*TP*CP*GP*CP*TP*GP*AP*TP*CP*CP*G)-3'
A: DNA POLYMERASE I
hetero molecules


Theoretical massNumber of molelcules
Total (without water)74,1227
Polymers73,4803
Non-polymers6424
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
E: 5'-D(*GP*GP*AP*TP*CP*AP*GP*CP*GP*A)-3'
F: 5'-D(*AP*CP*GP*TP*CP*GP*CP*TP*GP*AP*TP*CP*CP*G)-3'
B: DNA POLYMERASE I
hetero molecules


Theoretical massNumber of molelcules
Total (without water)74,0986
Polymers73,4803
Non-polymers6183
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)91.681, 91.681, 190.116
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number144
Space group name H-MP31

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Components

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DNA chain , 2 types, 4 molecules CEDF

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


Mass: 3094.043 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: PRIMER STRAND
#2: DNA chain 5'-D(*AP*CP*GP*TP*CP*GP*CP*TP*GP*AP*TP*CP*CP*G)-3'


Mass: 4256.767 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: Template Strand

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Protein , 1 types, 2 molecules AB

#3: Protein DNA POLYMERASE I /


Mass: 66128.836 Da / Num. of mol.: 2
Fragment: BACILLUS FRAGMENT (ANALOGOUS TO THE E. COLI KLENOW FRAGMENT)
Mutation: D329A
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).
Plasmid: pET-30A(+) / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)pLysS / References: UniProt: P52026, DNA-directed DNA polymerase

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Non-polymers , 5 types, 193 molecules

#4: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mn
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: SO4
#7: Chemical ChemComp-DCP / 2'-DEOXYCYTIDINE-5'-TRIPHOSPHATE / Deoxycytidine triphosphate


Mass: 467.157 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C9H16N3O13P3
#8: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 186 / 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: 3.12 Å3/Da / Density % sol: 60.63 %
Crystal growTemperature: 290 K / Method: vapor diffusion, hanging drop / pH: 5.8
Details: AMMONIUM SULFATE, MAGNESIUM SULFATE, MPD, MES, MANGANESE SULFATE, pH 5.80, VAPOR DIFFUSION, HANGING DROP, temperature 290.0K
Components of the solutions
IDNameCrystal-IDSol-ID
1AMMONIUM SULFATE11
2MgSO411
3MPD11
4MES11
5MnSo411
Crystal grow
*PLUS
pH: 5.8
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

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: NSLS / Beamline: X25 / Wavelength: 1.1 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Mar 29, 2001 / Details: MIRROR
RadiationMonochromator: Si Crystal / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.1 Å / Relative weight: 1
ReflectionResolution: 1.95→39.7 Å / Num. all: 129531 / Num. obs: 129531 / % possible obs: 99.8 % / Observed criterion σ(I): -3 / Redundancy: 4.9 % / Biso Wilson estimate: 23.2 Å2 / Rmerge(I) obs: 0.062 / Net I/σ(I): 18.6
Reflection shellResolution: 1.95→2.02 Å / Redundancy: 4.1 % / Rmerge(I) obs: 0.48 / Mean I/σ(I) obs: 2.5 / Num. unique all: 12800 / % possible all: 99.3
Reflection
*PLUS
Lowest resolution: 50 Å / % possible obs: 74.1 % / Num. measured all: 633019
Reflection shell
*PLUS
% possible obs: 39.8 % / Rmerge(I) obs: 0.48

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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: 1.95→39.7 Å / Rfactor Rfree error: 0.004 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 2 / Stereochemistry target values: Engh & Huber
Details: The crystal was twinned, with a partial twinning fraction of 0.485. Detwinning of the data was performed by treating the data as perfectly twinned, using the detwin_perfect.inp routine in ...Details: The crystal was twinned, with a partial twinning fraction of 0.485. Detwinning of the data was performed by treating the data as perfectly twinned, using the detwin_perfect.inp routine in CNS. The Bacillus polymerase was co-crystallized with ddATP, dCTP, and a DNA containing a 9 base pair duplex, a 4 base 5' template overhang, and a single base 3' template overhang. The 3' overhang on the template strand assured that the DNA duplex was not bound backwards by the polymerase during crystallization. ddATP was incorporated onto the 3' terminus of the primer strand. dCTP was not incorporated onto the primer strand, but was paired opposite the next template base. Thus, the resulting structure contained a 10 base pair DNA duplex with a 3' primer terminus and a 3 base 5' template overhang. Electron density was observed for all protein side chains except Lysine 298, which was modelled to the beta carbon. The manganese and magnesium atoms were assigned on the basis of low B-factor, proximity to coordinating residues, and by comparison to a related Klentaq polymerase structure (3KTQ). The data prevent a definitive assignment between MG 205 and water.
RfactorNum. reflection% reflectionSelection details
Rfree0.261 4574 4.7 %RANDOM
Rwork0.218 ---
all0.264 98839 --
obs0.218 96536 74.2 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 47.4519 Å2 / ksol: 0.364792 e/Å3
Displacement parametersBiso mean: 43.9 Å2
Baniso -1Baniso -2Baniso -3
1-0.3 Å20.74 Å20 Å2
2--0.3 Å20 Å2
3----0.6 Å2
Refine analyzeLuzzati coordinate error free: 0.32 Å / Luzzati sigma a free: 0.33 Å
Refinement stepCycle: LAST / Resolution: 1.95→39.7 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9294 974 69 186 10523
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.007
X-RAY DIFFRACTIONc_angle_deg1.3
X-RAY DIFFRACTIONc_dihedral_angle_d23.2
X-RAY DIFFRACTIONc_improper_angle_d1.42
X-RAY DIFFRACTIONc_mcbond_it0.661.5
X-RAY DIFFRACTIONc_mcangle_it1.122
X-RAY DIFFRACTIONc_scbond_it0.832
X-RAY DIFFRACTIONc_scangle_it1.262.5
LS refinement shellResolution: 1.95→2.02 Å / Rfactor Rfree error: 0.021 / Total num. of bins used: 10
RfactorNum. reflection% reflection
Rfree0.332 259 5 %
Rwork0.307 4895 -
obs--39.8 %
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.PARAMATER.TOP
X-RAY DIFFRACTION4ION.PARAMION.TOP
X-RAY DIFFRACTION5DCTP.PARAMDCTP.TOP
Refinement
*PLUS
Lowest resolution: 50 Å / % 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_deg23.2
X-RAY DIFFRACTIONc_improper_angle_d
X-RAY DIFFRACTIONc_improper_angle_deg1.42

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