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- EMDB-38837: The cryo-EM structure of TdpAB in complex with AMPPNP and PT-DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-38837
TitleThe cryo-EM structure of TdpAB in complex with AMPPNP and PT-DNA
Map data
Sample
  • Complex: TdpAB in complex with AMPPNP and PT-DNA
    • Protein or peptide: TdpA
    • Protein or peptide: TdpB
    • DNA: DNA (5'-D(P*GP*CP*CP*CP*TP*TP*TP*TP*GP*CP*AP*A)-3')
    • DNA: DNA (5'-D(P*TP*TP*GP*CP*AP*AP*AP*AP*GP*GP*GP*C)-3')
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
KeywordsDNA phosphorothioation / antiphage / translocase / nuclease / TRANSLOCASE-DNA complex
Biological speciesThermus antranikianii DSM 12462 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsChen TA / Qian T
Funding support China, 1 items
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
DepositionJan 25, 2024-
Header (metadata) releaseJan 29, 2025-
Map releaseJan 29, 2025-
UpdateJan 29, 2025-
Current statusJan 29, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_38837.png

Downloads & links

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Map

FileDownload / File: emd_38837.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 512 pix.
= 430.08 Å		0.84 Å/pix.
x 512 pix.
= 430.08 Å		0.84 Å/pix.
x 512 pix.
= 430.08 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.145
Minimum - Maximum-0.41720423 - 0.82655424
Average (Standard dev.)0.00025656825 (±0.022153081)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 430.08 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_38837_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_38837_half_map_2.map
Projections & Slices
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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : TdpAB in complex with AMPPNP and PT-DNA

EntireName: TdpAB in complex with AMPPNP and PT-DNA
Components
  • Complex: TdpAB in complex with AMPPNP and PT-DNA
    • Protein or peptide: TdpA
    • Protein or peptide: TdpB
    • DNA: DNA (5'-D(P*GP*CP*CP*CP*TP*TP*TP*TP*GP*CP*AP*A)-3')
    • DNA: DNA (5'-D(P*TP*TP*GP*CP*AP*AP*AP*AP*GP*GP*GP*C)-3')
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

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Supramolecule #1: TdpAB in complex with AMPPNP and PT-DNA

SupramoleculeName: TdpAB in complex with AMPPNP and PT-DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Thermus antranikianii DSM 12462 (bacteria)

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Macromolecule #1: TdpA

MacromoleculeName: TdpA / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Thermus antranikianii DSM 12462 (bacteria)
Molecular weightTheoretical: 66.546008 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAESPIGVVV SSRRNGPWAE LTLVLTPQEL DQGKRLLLGE LVRVSSGGKD YVGMVLDGYY EPVGRSDPTY TLALAHINQV DLEKEDPWA RKEVNFYHHR IVLLGRVVQG GLFAPSTRLL PPVVEARVYR MTEEELQRLL AAEVRTSGSV KAEGKRRYAF G HLAYGLEE ...String:
MAESPIGVVV SSRRNGPWAE LTLVLTPQEL DQGKRLLLGE LVRVSSGGKD YVGMVLDGYY EPVGRSDPTY TLALAHINQV DLEKEDPWA RKEVNFYHHR IVLLGRVVQG GLFAPSTRLL PPVVEARVYR MTEEELQRLL AAEVRTSGSV KAEGKRRYAF G HLAYGLEE GGEYPEVVKE VDPALFVGRR TANFGKTGFG KSNENKVILT LLAHAFPRVG MLILDQNAEY LLQTEATTSP GL AQAFKAL GIRGRIRFYT AREEAWARRL KEHLGTEWRE YVEVLPLKVD FYHFPELAVA LAYQRRRLQG AEPPQYLENA FYN LEDWKH IPDRMAYVYG ALRKAGLTPR KGLKIKYKNE NYDISEEKSW GNLQEAMENN SQRGDNKGGA RELYSRAKVF SFLR AFHAP GKEANFLETI KEDLLGEKTE GEGKVVILDL PSLGEAADFF TLRLMDLLFD RAVELYGKRQ ANFLVVLEEA HNFLE DKAG IFYRVAKEGR KYGIGMLYST QSPASIPMEI LSQTENFLVK HLSSEEDVKV LKRAKAPFAF VADFLLSEPI IGYSYV YFE PYQPFVVPLR VKLLEHVLKS LDS

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Macromolecule #2: TdpB

MacromoleculeName: TdpB / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Thermus antranikianii DSM 12462 (bacteria)
Molecular weightTheoretical: 42.757934 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MPYAGEGSNP LGLKDFLDDL RLDHYQDLLR ELDELYQKLK QERQVPLHGD GEAYPLLTLT VDGGEGRAFE ELPLLSFGLV RVAAVGVKG FRLPSIAHLL PGYEVLRDPK GYLEGLLERS EESPAADALK TFFRATGISL EDLGEYYTKD LRAFMGIFRD V LEWAYLVW ...String:
MPYAGEGSNP LGLKDFLDDL RLDHYQDLLR ELDELYQKLK QERQVPLHGD GEAYPLLTLT VDGGEGRAFE ELPLLSFGLV RVAAVGVKG FRLPSIAHLL PGYEVLRDPK GYLEGLLERS EESPAADALK TFFRATGISL EDLGEYYTKD LRAFMGIFRD V LEWAYLVW GVEKVLQESY KDYLFIKDGR LAQLGVRESF RSKLQNYFAR KHLLLAGVTK RSRLLAEGLT SLVMARLFAE AR GTFVLQV PQELMEKAYR YERQWNADLE GAFVMGRRYV ARLLEDTFRP QEGVAIFDLP PYLGEEDAVK VARSLRAHRS VLY GGSVGT VVEAHGRASV ARSIPRRMEE EILARFRKAF GEDLAKKLTE WLRLADRED

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Macromolecule #3: DNA (5'-D(P*GP*CP*CP*CP*TP*TP*TP*TP*GP*CP*AP*A)-3')

MacromoleculeName: DNA (5'-D(P*GP*CP*CP*CP*TP*TP*TP*TP*GP*CP*AP*A)-3') / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Thermus antranikianii DSM 12462 (bacteria)
Molecular weightTheoretical: 3.613366 KDa
SequenceString:
(DG)(DC)(DC)(DC)(DT)(DT)(DT)(DT)(DG)(DC) (DA)(DA)

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Macromolecule #4: DNA (5'-D(P*TP*TP*GP*CP*AP*AP*AP*AP*GP*GP*GP*C)-3')

MacromoleculeName: DNA (5'-D(P*TP*TP*GP*CP*AP*AP*AP*AP*GP*GP*GP*C)-3') / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Thermus antranikianii DSM 12462 (bacteria)
Molecular weightTheoretical: 3.711442 KDa
SequenceString:
(DT)(DT)(DG)(DC)(DA)(DA)(DA)(DA)(DG)(DG) (DG)(DC)

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Macromolecule #5: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 5 / Number of copies: 3 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 1.3 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 78868
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE

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