3QEI

RB69 DNA Polymerase (L561A/S565G/Y567A) Ternary Complex with dCTP Opposite Difluorotoluene Nucleoside


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.18 Å
  • R-Value Free: 0.238 
  • R-Value Work: 0.190 
  • R-Value Observed: 0.193 

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This is version 1.3 of the entry. See complete history


Literature

Structural Basis for Differential Insertion Kinetics of dNMPs Opposite a Difluorotoluene Nucleotide Residue.

Xia, S.Eom, S.H.Konigsberg, W.H.Wang, J.

(2012) Biochemistry 51: 1476-1485

  • DOI: https://doi.org/10.1021/bi2016487
  • Primary Citation of Related Structures:  
    3QEI, 3QER, 3QES

  • PubMed Abstract: 

    We have recently challenged the widely held view that 2,4-difluorotoluene (dF) is a nonpolar isosteric analogue of the nucleotide dT, incapable of forming hydrogen bonds (HBs). To gain a further understanding for the kinetic preference that favors dAMP insertion opposite a templating dF, a result that mirrors the base selectivity that favors dAMP insertion opposite dT by RB69 DNA polymerase (RB69pol), we determined presteady-state kinetic parameters for incorporation of four dNMPs opposite dF by RB69pol and solved the structures of corresponding ternary complexes. We observed that both the F2 and F4 substituent of dF in these structures serve as HB acceptors forming HBs either directly with dTTP and dGTP or indirectly with dATP and dCTP via ordered water molecules. We have defined the shape and chemical features of each dF/dNTP pair in the RB69pol active site without the corresponding phosphodiester-linkage constraints of dF/dNs when they are embedded in isolated DNA duplexes. These features can explain the kinetic preferences exhibited by the templating dF when the nucleotide incorporation is catalyzed by wild type RB69pol or its mutants. We further show that the shapes of the dNTP/dF nascent base pair differ markedly from the corresponding dNTP/dT in the pol active site and that these differences have a profound effect on their incorporation efficiencies.


  • Organizational Affiliation

    Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114, United States.


Macromolecules

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Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
DNA polymerase903Escherichia phage RB69Mutation(s): 3 
Gene Names: 43
EC: 2.7.7.7
UniProt
Find proteins for Q38087 (Escherichia phage RB69)
Explore Q38087 
Go to UniProtKB:  Q38087
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ38087
Sequence Annotations
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  • Reference Sequence

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Entity ID: 2
MoleculeChains LengthOrganismImage
DNA (5'-D(P*TP*CP*GP*(DFT)P*GP*TP*AP*AP*GP*CP*AP*GP*TP*CP*CP*GP*CP*G)-3')B [auth T]18synthetic construct
Sequence Annotations
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  • Reference Sequence

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Entity ID: 3
MoleculeChains LengthOrganismImage
DNA (5'-D(*GP*CP*GP*GP*AP*CP*TP*GP*CP*TP*TP*AP*(DOC))-3')C [auth P]13synthetic construct
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.18 Å
  • R-Value Free: 0.238 
  • R-Value Work: 0.190 
  • R-Value Observed: 0.193 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 78.345α = 90
b = 118.231β = 90
c = 130.854γ = 90
Software Package:
Software NamePurpose
HKL-2000data collection
AMoREphasing
REFMACrefinement
HKL-2000data reduction
HKL-2000data scaling

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2012-01-25
    Type: Initial release
  • Version 1.1: 2012-02-22
    Changes: Database references
  • Version 1.2: 2012-03-14
    Changes: Database references
  • Version 1.3: 2024-02-21
    Changes: Data collection, Database references, Derived calculations