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- PDB-8wfd: The cryo-EM structure of TdpAB in complex with AMPPNP and DNA -

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Basic information

Entry
Database: PDB / ID: 8wfd
TitleThe cryo-EM structure of TdpAB in complex with AMPPNP and DNA
Components
  • DNA (5'-D(P*GP*CP*CP*CP*TP*TP*TP*TP*GP*CP*AP*A)-3')
  • DNA (5'-D(P*TP*TP*GP*CP*AP*AP*AP*AP*GP*GP*GP*C)-3')
  • TdpA
  • TdpB
KeywordsTRANSLOCASE/DNA / DNA phosphorothioation / antiphage / translocase / nuclease / TRANSLOCASE-DNA complex
Function / homologyPHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / DNA / DNA (> 10)
Function and homology information
Biological speciesThermus antranikianii DSM 12462 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.67 Å
AuthorsAn, T. / Tan, Q.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Nat Chem Biol / Year: 2025
Title: A DNA phosphorothioation pathway via adenylated intermediate modulates Tdp machinery.
Authors: Tianchen An / Qian Tan / Lixu Jiang / Li Liu / Xing Jiang / Liying Liu / Xiaofei Chang / Xihao Tian / Zixin Deng / Shuai Gao / Lianrong Wang / Shi Chen /
Abstract: In prokaryotes, the non-bridging oxygen in the DNA sugar-phosphate backbone can be enzymatically replaced by a sulfur atom, resulting in phosphorothioate (PT) modification. However, the mechanism ...In prokaryotes, the non-bridging oxygen in the DNA sugar-phosphate backbone can be enzymatically replaced by a sulfur atom, resulting in phosphorothioate (PT) modification. However, the mechanism underlying the oxygen-to-sulfur substitution remains enigmatic. In this study, we discovered a hypercompact DNA phosphorothioation system, TdpABC, in extreme thermophiles. This DNA sulfuration process occurs through two sequential steps: an initial activation step by ATP to form an adenylated intermediate, followed by a substitution step where the adenyl group is replaced with a sulfur atom. Together with the TdpA-TdpB, the TdpABC system provides anti-phage defense by degrading PT-free phage DNA. Cryogenic electron microscopy structural analysis revealed that the TdpA hexamer binds one strand of encircled duplex DNA via hydrogen bonds arranged in a spiral staircase conformation. Nevertheless, the TdpAB-DNA interaction was sensitive to the hydrophobicity of the PT sulfur. PTs inhibit ATP-driven translocation and nuclease activity of TdpAB on self-DNA, thereby preventing autoimmunity.
History
DepositionSep 19, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 22, 2025Provider: repository / Type: Initial release
Revision 1.1Jan 29, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.pdbx_database_id_PubMed / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
F: TdpA
A: TdpA
B: TdpA
E: TdpA
D: TdpA
C: TdpA
H: TdpB
G: TdpB
I: DNA (5'-D(P*GP*CP*CP*CP*TP*TP*TP*TP*GP*CP*AP*A)-3')
J: DNA (5'-D(P*TP*TP*GP*CP*AP*AP*AP*AP*GP*GP*GP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)493,63513
Polymers492,11710
Non-polymers1,5193
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
TdpA


Mass: 66546.008 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus antranikianii DSM 12462 (bacteria)
Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Protein TdpB


Mass: 42757.934 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus antranikianii DSM 12462 (bacteria)
Production host: Escherichia coli BL21(DE3) (bacteria)
#3: DNA chain DNA (5'-D(P*GP*CP*CP*CP*TP*TP*TP*TP*GP*CP*AP*A)-3')


Mass: 3613.366 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Thermus antranikianii DSM 12462 (bacteria)
#4: DNA chain DNA (5'-D(P*TP*TP*GP*CP*AP*AP*AP*AP*GP*GP*GP*C)-3')


Mass: 3711.442 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Thermus antranikianii DSM 12462 (bacteria)
#5: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP-PNP, energy-carrying molecule analogue*YM
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: TdpAB in complex with AMPPNP and DNA / Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Source (natural)Organism: Thermus antranikianii DSM 12462 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 1300 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 2.67 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 223498 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 36.57 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004134428
ELECTRON MICROSCOPYf_angle_d0.468746656
ELECTRON MICROSCOPYf_chiral_restr0.04095038
ELECTRON MICROSCOPYf_plane_restr0.00375936
ELECTRON MICROSCOPYf_dihedral_angle_d8.98544905

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