9FM3

KlenTaq DNA polymerase in a ternary complex with primer/template and a selenophene-modified dUTP (SedUTP)

PDB DOI: https://doi.org/10.2210/pdb9FM3/pdb

Classification: TRANSFERASE
Organism(s): Thermus aquaticus, synthetic construct
Expression System: Escherichia coli
Mutation(s): No

Deposited: 2024-06-05 Released: 2024-11-13
Deposition Author(s): Betz, K., Srivatsan, S.G.
Funding Organization(s): Not funded

Experimental Data Snapshot

Method: X-RAY DIFFRACTION
Resolution: 2.59 Å
R-Value Free: 0.245
R-Value Work: 0.187
R-Value Observed: 0.190

Starting Model: experimental
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wwPDB Validation 3D Report Full Report

Ligand Structure Quality Assessment

This is version 1.0 of the entry. See complete history.

Literature

Structures of a DNA Polymerase Caught while Incorporating Responsive Dual-Functional Nucleotide Probes.

Ghosh, P., Betz, K., Gutfreund, C., Pal, A., Marx, A., Srivatsan, S.G.

(2024) Angew Chem Int Ed Engl : e202414319-e202414319

PubMed: 39428682 Search on PubMed
DOI: https://doi.org/10.1002/anie.202414319
Primary Citation of Related Structures:
9FM3, 9FMF

PubMed Abstract:
Functionalizing nucleic acids using DNA polymerases is essential in biophysical and biotechnology applications. This study focuses on understanding how DNA polymerases recognize and incorporate nucleotides with diverse chemical modifications, aiming to develop advanced nucleotide probes. We present the crystal structures of ternary complexes of Thermus aquaticus DNA polymerase (KlenTaq) with C5-heterocycle-modified environment-sensitive 2'-deoxyuridine-5'-triphosphate (dUTP) probes. These nucleotides include SedUTP, BFdUTP and FBFdUTP, which bear selenophene, benzofuran and fluorobenzofuran, respectively, at the C5 position of uracil, and exhibit high conformational sensitivity. SedUTP and FBFdUTP serve as dual-app probes, combining a fluorophore with X-ray anomalous scattering Se or 19F NMR labels. Our study reveals that the size of the heterocycle influences how DNA polymerase families A and B incorporate these modified nucleotides during single nucleotide incorporation and primer extension reactions. Remarkably, FBFdUTP's responsiveness enabled real-time monitoring of the binary complex formation and polymerase activity through fluorescence and 19F NMR. Comparative analysis of incorporation profiles, fluorescence, 19F NMR data, and crystal structures of ternary complexes highlights the enzyme's plasticity. Key insights are provided into the role of gatekeeper amino acids (Arg660 and Arg587) in accommodating and processing these modified substrates, offering a structural basis for next-generation nucleotide probe development.

Organizational Affiliation

Indian Institute of Science Education and Research Pune, Chemistry, Dr. Homi Bhabha Road, 411008, Pune, INDIA.

Macromolecule Content

Total Structure Weight: 70.12 kDa
Atom Count: 4,903
Modelled Residue Count: 567
Deposited Residue Count: 567
Unique protein chains: 1
Unique nucleic acid chains: 2

Macromolecules

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(by identity cutoff) | 3D Structure

Entity ID: 1
Molecule	Chains	Sequence Length	Organism	Details	Image
DNA polymerase I, thermostable	A	540	Thermus aquaticus	Mutation(s): 0 Gene Names: polA, pol1 EC: 2.7.7.7
UniProt
Find proteins for P19821 (Thermus aquaticus) Explore P19821 Go to UniProtKB: P19821
Entity Groups
Sequence Clusters	30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt Group	P19821
Sequence Annotations Expand
Reference Sequence

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Entity ID: 2
Molecule	Chains	Length	Organism	Image
Primer with terminal DOC	B	11	synthetic construct
Sequence Annotations Expand
Reference Sequence

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Entity ID: 3
Molecule	Chains	Length	Organism	Image
DNA template	C	16	synthetic construct
Sequence Annotations Expand
Reference Sequence

Small Molecules

Ligands 3 Unique
ID	Chains	Name / Formula / InChI Key	2D Diagram	3D Interactions
A1IDZ (Subject of Investigation/LOI) Query on A1IDZ Download Ideal Coordinates CCD File SDF format, chain D [auth A] MOL2 format, chain D [auth A] mmCIF format, chain D [auth A]	D [auth A]	[[(2~{R},3~{S},5~{R})-5-[2,4-bis(oxidanylidene)-5-selenophen-2-yl-pyrimidin-1-yl]-3-oxidanyl-oxolan-2-yl]methoxy-oxidanyl-phosphoryl] phosphono hydrogen phosphate C₁₃ H₁₇ N₂ O₁₄ P₃ Se IFHZFLUXRCNTJE-QCZWPSDZSA-N		Interactions Focus chain D [auth A] Interactions & Density Focus chain D [auth A]
DOC (Subject of Investigation/LOI) Query on DOC Download Ideal Coordinates CCD File SDF format, chain F [auth B] MOL2 format, chain F [auth B] mmCIF format, chain F [auth B]	F [auth B]	2',3'-DIDEOXYCYTIDINE-5'-MONOPHOSPHATE C₉ H₁₄ N₃ O₆ P RAJMXAZJKUGYGW-POYBYMJQSA-N		Interactions Focus chain F [auth B] Interactions & Density Focus chain F [auth B]
MG (Subject of Investigation/LOI) Query on MG Download Ideal Coordinates CCD File SDF format, chain E [auth A] MOL2 format, chain E [auth A] mmCIF format, chain E [auth A]	E [auth A]	MAGNESIUM ION Mg JLVVSXFLKOJNIY-UHFFFAOYSA-N		Interactions Focus chain E [auth A] Interactions & Density Focus chain E [auth A]