5U9H

DNA polymerase beta product complex with inserted Sp-isomer of dCTP-alpha-S


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.85 Å
  • R-Value Free: 0.233 
  • R-Value Work: 0.195 
  • R-Value Observed: 0.196 

wwPDB Validation   3D Report Full Report


This is version 1.4 of the entry. See complete history


Literature

Revealing the role of the product metal in DNA polymerase beta catalysis.

Perera, L.Freudenthal, B.D.Beard, W.A.Pedersen, L.G.Wilson, S.H.

(2017) Nucleic Acids Res 45: 2736-2745

  • DOI: https://doi.org/10.1093/nar/gkw1363
  • Primary Citation of Related Structures:  
    5U9H

  • PubMed Abstract: 

    DNA polymerases catalyze a metal-dependent nucleotidyl transferase reaction during extension of a DNA strand using the complementary strand as a template. The reaction has long been considered to require two magnesium ions. Recently, a third active site magnesium ion was identified in some DNA polymerase product crystallographic structures, but its role is not known. Using quantum mechanical/ molecular mechanical calculations of polymerase β, we find that a third magnesium ion positioned near the newly identified product metal site does not alter the activation barrier for the chemical reaction indicating that it does not have a role in the forward reaction. This is consistent with time-lapse crystallographic structures following insertion of Sp-dCTPαS. Although sulfur substitution deters product metal binding, this has only a minimal effect on the rate of the forward reaction. Surprisingly, monovalent sodium or ammonium ions, positioned in the product metal site, lowered the activation barrier. These calculations highlight the impact that an active site water network can have on the energetics of the forward reaction and how metals or enzyme side chains may interact with the network to modulate the reaction barrier. These results also are discussed in the context of earlier findings indicating that magnesium at the product metal position blocks the reverse pyrophosphorolysis reaction.


  • Organizational Affiliation

    Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233, Research Triangle Park, NC 27709-2233, USA.


Macromolecules

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Entity ID: 4
MoleculeChains Sequence LengthOrganismDetailsImage
DNA polymerase betaD [auth A]335Homo sapiensMutation(s): 0 
Gene Names: POLB
EC: 2.7.7.7 (PDB Primary Data), 4.2.99 (PDB Primary Data)
UniProt & NIH Common Fund Data Resources
Find proteins for P06746 (Homo sapiens)
Explore P06746 
Go to UniProtKB:  P06746
PHAROS:  P06746
GTEx:  ENSG00000070501 
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP06746
Sequence Annotations
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  • Reference Sequence

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Entity ID: 1
MoleculeChains LengthOrganismImage
DNA (5'-D(*CP*CP*GP*AP*CP*GP*GP*CP*GP*CP*AP*TP*CP*AP*GP*C)-3')A [auth T]16synthetic construct
Sequence Annotations
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  • Reference Sequence

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Entity ID: 2
MoleculeChains LengthOrganismImage
DNA (5'-D(*GP*CP*TP*GP*AP*TP*GP*CP*GP*CP*(C7R))-3')B [auth P]11synthetic construct
Sequence Annotations
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  • Reference Sequence

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

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.85 Å
  • R-Value Free: 0.233 
  • R-Value Work: 0.195 
  • R-Value Observed: 0.196 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 50.98α = 90
b = 80.048β = 108.05
c = 55.074γ = 90
Software Package:
Software NamePurpose
PHENIXrefinement
HKL-2000data reduction
HKL-2000data scaling
PHENIXphasing

Structure Validation

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Entry History & Funding Information

Deposition Data


Funding OrganizationLocationGrant Number
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)United States--

Revision History  (Full details and data files)

  • Version 1.0: 2017-02-08
    Type: Initial release
  • Version 1.1: 2017-05-03
    Changes: Database references
  • Version 1.2: 2017-09-27
    Changes: Author supporting evidence
  • Version 1.3: 2019-12-04
    Changes: Author supporting evidence
  • Version 1.4: 2023-10-04
    Changes: Data collection, Database references, Derived calculations, Refinement description