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- PDB-2ajq: Structure of replicative DNA polymerase provides insigts into the... -

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Basic information

Entry
Database: PDB / ID: 2ajq
TitleStructure of replicative DNA polymerase provides insigts into the mechanisms for processivity, frameshifting and editing
Components
  • DNA PrimerPrimer (molecular biology)
  • DNA Template
  • T7 DNA polymerase
  • thioredoxin 1
KeywordsTRANSFERASE / TRANSCRIPTION/DNA / Polymerase T7 / x-ray crystallography / ternary complex / TRANSCRIPTION-DNA COMPLEX
Function / homology
Function and homology information


DNA synthesis involved in DNA replication / DNA exonuclease activity / viral DNA genome replication / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA polymerase processivity factor activity / protein-disulfide reductase activity / 3'-5' exonuclease activity / cell redox homeostasis / DNA-templated DNA replication / DNA-directed DNA polymerase ...DNA synthesis involved in DNA replication / DNA exonuclease activity / viral DNA genome replication / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA polymerase processivity factor activity / protein-disulfide reductase activity / 3'-5' exonuclease activity / cell redox homeostasis / DNA-templated DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / DNA binding / metal ion binding / cytosol / cytoplasm
Similarity search - Function
DNA-directed DNA polymerase T7 / Taq DNA Polymerase; Chain T, domain 4 / Taq DNA Polymerase; Chain T, domain 4 / Alpha-Beta Plaits - #370 / Thioredoxin / 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-directed DNA polymerase T7 / Taq DNA Polymerase; Chain T, domain 4 / Taq DNA Polymerase; Chain T, domain 4 / Alpha-Beta Plaits - #370 / Thioredoxin / 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 / Thioredoxin / Thioredoxin, conserved site / Thioredoxin family active site. / Thioredoxin domain profile. / Thioredoxin domain / 5' to 3' exonuclease, C-terminal subdomain / Ribonuclease H-like superfamily/Ribonuclease H / Glutaredoxin / Glutaredoxin / DNA polymerase; domain 1 / Nucleotidyltransferase; domain 5 / Thioredoxin-like superfamily / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Alpha-Beta Plaits / DNA/RNA polymerase superfamily / Up-down Bundle / 2-Layer Sandwich / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA-directed DNA polymerase / Thioredoxin 1
Similarity search - Component
Biological speciesEnterobacteria phage T7 (virus)
Escherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.6 Å
AuthorsBrieba, L. / Ellenberger, T.
CitationJournal: To be Published
Title: Structure of replicative DNA polymerase provides insigts into the mechanisms for processivity, frameshifting and editing
Authors: Brieba, L. / Ellenberger, T.
History
DepositionAug 2, 2005Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 26, 2006Provider: repository / Type: Initial release
Revision 1.1Apr 30, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Oct 20, 2021Group: Database references / Derived calculations / Category: database_2 / struct_conn / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_leaving_atom_flag / _struct_ref_seq_dif.details
Revision 1.4Feb 14, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
P: DNA Primer
T: DNA Template
X: DNA Primer
Z: DNA Template
A: T7 DNA polymerase
B: thioredoxin 1
F: T7 DNA polymerase
I: thioredoxin 1


Theoretical massNumber of molelcules
Total (without water)212,3138
Polymers212,3138
Non-polymers00
Water7,458414
1
P: DNA Primer
T: DNA Template
A: T7 DNA polymerase
B: thioredoxin 1


Theoretical massNumber of molelcules
Total (without water)106,1564
Polymers106,1564
Non-polymers00
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
X: DNA Primer
Z: DNA Template
F: T7 DNA polymerase
I: thioredoxin 1


Theoretical massNumber of molelcules
Total (without water)106,1564
Polymers106,1564
Non-polymers00
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)168.312, 169.235, 179.788
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number20
Cell settingorthorhombic
Space group name H-MC2221

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Components

#1: DNA chain DNA Primer / Primer (molecular biology)


Mass: 6739.384 Da / Num. of mol.: 2 / Source method: obtained synthetically
#2: DNA chain DNA Template


Mass: 8026.148 Da / Num. of mol.: 2 / Source method: obtained synthetically
#3: Protein T7 DNA polymerase / / E.C.2.7.7.7


Mass: 79703.578 Da / Num. of mol.: 2 / Mutation: Residues 5 and 7 mutated to Ala
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterobacteria phage T7 (virus) / Genus: T7-like viruses
Description: Protein induced at an OD_600 of 0.5 by addition of 0.5 mM IPTG for 4 hours at 37C
Gene: 5 / Plasmid: pGP5 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 (DE3) pLys S / References: UniProt: P00581, DNA-directed DNA polymerase
#4: Protein thioredoxin 1 / / TRX1 / TRX


Mass: 11687.388 Da / Num. of mol.: 2 / Mutation: Residues 5 and 7 mutated to Ala
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Description: Protein induced at an OD_600 of 0.5 by addition of 0.5 mM IPTG for 4 hours at 37C
Gene: trxA, fipA, tsnC / Plasmid: p-Trx / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 (DE3) pLys S / References: UniProt: P0AA25
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 414 / 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: 3.18 Å3/Da / Density % sol: 61 %
Crystal growTemperature: 273 K / Method: vapor diffusion, hanging drop / pH: 7.5
Details: A complex of 1x10^-4 M T7 DNA polymerase 5A7A:thioredoxin was assembled with an equimolar amount of double stranded DNA substrate. Crystallization was achieved using a buffer containing 50mM ...Details: A complex of 1x10^-4 M T7 DNA polymerase 5A7A:thioredoxin was assembled with an equimolar amount of double stranded DNA substrate. Crystallization was achieved using a buffer containing 50mM HEPES pH 7.5, 10mM MgCl_2, 2mM DTT, and 0.5 mM terminal ddTTP Seed crystals were grown by hanging drop vapor diffusion by mixing 1ul each of protein-DNA solution and a reservoir solutions containing between 16 to 20% PEG 8000, 100mM ACES pH 7.5, 120 ammonium sulfate, 30mM MgCl2, and 5mM DTT. These crystals were used to streak-seed a grid of protein/reservoir solutions with concentrations of PEG 8000 between 13 to 15%. Pyramidal crystals appeared overnight and reached a maximum size of ~150 X 150 X 100 um3 after 3 to 4 days. Crystals were harvested overnight in mother-liquor containing 10 % PEG 400, temperature 273K, VAPOR DIFFUSION, HANGING DROP
Components of the solutions
IDNameCrystal-IDSol-ID
1HEPES11
2MgCl211
3DTT11
4ddTTP11
5PEG 800011
6ACES11
7(NH4)2SO411
8HEPES12
9MgCl212
10DTT1
11ddTTP12
12PEG 800012
13ACES12
14(NH4)2SO412

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: NSLS / Beamline: X26C / Wavelength: 1.1 / Wavelength: 1.1 Å
DetectorType: ADSC QUANTUM 4 / Detector: CCD / Date: Mar 28, 2005 / Details: mirrors
RadiationMonochromator: graphite / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.1 Å / Relative weight: 1
ReflectionResolution: 2.38→45.97 Å / Num. all: 98459 / Num. obs: 76808 / % possible obs: 99.8 % / Observed criterion σ(I): 3 / Redundancy: 7.7 % / Biso Wilson estimate: 39.2 Å2 / Rmerge(I) obs: 0.087 / Rsym value: 0.111 / Net I/σ(I): 16.8
Reflection shellHighest resolution: 2.38 Å / Rmerge(I) obs: 0.333 / Mean I/σ(I) obs: 5.8 / % possible all: 100

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Processing

Software
NameVersionClassification
HKL-2000data collection
TRUNCATEdata reduction
CNSrefinement
HKL-2000data reduction
CCP4(TRUNCATE)data scaling
CNSphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.6→45.97 Å / Rfactor Rfree error: 0.005 / Data cutoff high absF: 4557696.91 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0
RfactorNum. reflection% reflectionSelection details
Rfree0.284 3929 5 %RANDOM
Rwork0.233 ---
obs0.2331 78685 99.8 %-
all-78685 --
Solvent computationSolvent model: FLAT MODEL / Bsol: 46.0629 Å2 / ksol: 0.315331 e/Å3
Displacement parametersBiso mean: 74.2 Å2
Baniso -1Baniso -2Baniso -3
1--7.72 Å20 Å20 Å2
2---8.54 Å20 Å2
3---16.26 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.45 Å0.35 Å
Luzzati d res low-5 Å
Luzzati sigma a0.48 Å0.34 Å
Refinement stepCycle: LAST / Resolution: 2.6→45.97 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms12588 1789 0 414 14791
Refine LS restraints
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONc_bond_d0.007
X-RAY DIFFRACTIONc_angle_deg1.2
X-RAY DIFFRACTIONc_dihedral_angle_d21.5
X-RAY DIFFRACTIONc_improper_angle_d0.98
LS refinement shellResolution: 2.6→2.76 Å / Rfactor Rfree error: 0.016 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.388 621 4.8 %
Rwork0.305 12355 -
obs--100 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1protein_rep.paramprotein.top
X-RAY DIFFRACTION2dna-rna-tt.param
X-RAY DIFFRACTION3ion.param
X-RAY DIFFRACTION4water.param

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