+Open data
-Basic information
Entry | Database: PDB / ID: 1njw | |||||||||
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Title | GUANINE-THYMINE MISMATCH AT THE POLYMERASE ACTIVE SITE | |||||||||
Components |
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Keywords | TRANSFERASE/DNA / DNA POLYMERASE I / DNA REPLICATION / KLENOW FRAGMENT / PROTEIN-DNA COMPLEX / DNA MISMATCH / 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 / nucleotide binding / DNA repair / DNA binding / metal ion binding Similarity search - Function | |||||||||
Biological species | Geobacillus stearothermophilus (bacteria) | |||||||||
Method | X-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 1.9 Å | |||||||||
Authors | Johnson, 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 |
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Remark 6 | THE POLYMERASE-DNA COMPLEX ADOPTS A DISTORTED OPEN CONFORMATION WITH A GUANINE-THYMINE MISMATCH ... THE POLYMERASE-DNA COMPLEX ADOPTS A DISTORTED OPEN CONFORMATION WITH A GUANINE-THYMINE MISMATCH BOUND AT THE POLYMERASE POST-INSERTION SITE. THE MISMATCH ADOPTS A WOBBLE CONFORMATION. | |||||||||
Remark 999 | SEQUENCE 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. |
-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 1njw.cif.gz | 158.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb1njw.ent.gz | 116.3 KB | Display | PDB format |
PDBx/mmJSON format | 1njw.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/nj/1njw ftp://data.pdbj.org/pub/pdb/validation_reports/nj/1njw | HTTPS FTP |
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-Related structure data
Related structure data | 1njxC 1njyC 1njzC 1nk0C 1nk4C 1nk5C 1nk6C 1nk7C 1nk8C 1nk9C 1nkbC 1nkcC 1nkeC 2bdpS C: citing same article (ref.) S: Starting model for refinement |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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Unit cell |
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Details | Exists as a monomer. One molecule per asymmetric unit. |
-Components
-DNA chain , 2 types, 2 molecules BC
#1: DNA chain | Mass: 3399.223 Da / Num. of mol.: 1 / Source method: obtained synthetically |
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#2: DNA chain | Mass: 4569.973 Da / Num. of mol.: 1 / Source method: obtained synthetically |
-Protein / Sugars , 2 types, 3 molecules A
#3: Protein | 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). Plasmid: PET-30A(+) / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)PLYSS References: UniProt: Q5KWC1*PLUS, DNA-directed DNA polymerase |
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#4: Polysaccharide |
-Non-polymers , 3 types, 432 molecules
#5: Chemical | #6: Chemical | ChemComp-MG / | #7: Water | ChemComp-HOH / | |
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-Experimental details
-Experiment
Experiment | Method: X-RAY DIFFRACTION / Number of used crystals: 1 |
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-Sample preparation
Crystal | Density Matthews: 2.75 Å3/Da / Density % sol: 54.98 % | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Crystal grow | Temperature: 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 |
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Crystal grow | *PLUS pH: 5.8 / Method: vapor diffusion, hanging dropDetails: Johnson, S.J., (2003) Proc.Natl.Acad.Sci.USA, 100, 3895. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Components of the solutions | *PLUS
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-Data collection
Diffraction |
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Diffraction source | Source: ROTATING ANODE / Type: RIGAKU RU200 / Wavelength: 1.5418 | |||||||||
Detector | Type: RIGAKU RAXIS IV / Detector: IMAGE PLATE / Date: Dec 23, 2000 / Details: mirrors | |||||||||
Radiation | Monochromator: NI FILTER / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray | |||||||||
Radiation wavelength | Wavelength: 1.5418 Å / Relative weight: 1 | |||||||||
Reflection | Resolution: 1.9→32.48 Å / Num. all: 63840 / Num. obs: 63840 / % possible obs: 92.5 % / Observed criterion σ(F): -3 / Observed criterion σ(I): -3 / Redundancy: 4.9 % / Biso Wilson estimate: 19.7 Å2 / Rsym value: 0.059 / Net I/σ(I): 23.4 | |||||||||
Reflection shell | Resolution: 1.9→1.97 Å / Redundancy: 3.8 % / Mean I/σ(I) obs: 3.8 / Num. unique all: 4179 / Rsym value: 0.366 / % possible all: 61.6 | |||||||||
Reflection | *PLUS Lowest resolution: 50 Å / Num. obs: 63846 / Num. measured all: 311125 / Rmerge(I) obs: 0.059 | |||||||||
Reflection shell | *PLUS Highest resolution: 1.9 Å / Rmerge(I) obs: 0.366 |
-Processing
Software |
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Refinement | Method to determine structure: MOLECULAR REPLACEMENT Starting model: PDB ENTRY 2BDP Resolution: 1.9→32.48 Å / Rfactor Rfree error: 0.004 / Data cutoff high absF: 2320550.25 / Data cutoff high rms absF: 2320550.25 / 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 5 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 5 BASE 5' TEMPLATE OVERHANG. THE CRYSTAL WAS SUBSEQUENTLY PLACED INTO A STABILIZING SOLUTION CONTAINING DGTP AND MANGANESE SULFATE. THE RESULTING PRODUCT COMPLEX CONTAINED 11 BASE PAIRS OF DUPLEX DNA AND A 3 BASE 5' TEMPLATE OVERHANG. A GUANINE-THYMINE MISMATCH IS LOCATED AT THE 3' PRIMER TERMINUS OF THE DNA DUPLEX. THE 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 298, 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.
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Solvent computation | Solvent model: FLAT MODEL / Bsol: 40.848 Å2 / ksol: 0.37735 e/Å3 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 30.6 Å2
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Refine analyze |
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Refinement step | Cycle: LAST / Resolution: 1.9→32.48 Å
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Refine LS restraints |
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LS refinement shell | Resolution: 1.9→1.97 Å / Rfactor Rfree error: 0.022 / Total num. of bins used: 10
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Xplor file |
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Refinement | *PLUS Highest resolution: 1.97 Å / Lowest resolution: 50 Å / % reflection Rfree: 5 % / Rfactor Rfree: 0.243 / Rfactor Rwork: 0.213 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Solvent computation | *PLUS | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | *PLUS | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints | *PLUS
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LS refinement shell | *PLUS Rfactor Rfree: 0.33 / Rfactor Rwork: 0.271 |