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- PDB-9jo8: Cryo-EM structure of the mono-DdCBE bound to a dsDNA substrate. -

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Basic information

Entry
Database: PDB / ID: 9jo8
TitleCryo-EM structure of the mono-DdCBE bound to a dsDNA substrate.
Components
  • (TALE repeat protein ...) x 2
  • Double-stranded DNA deaminase toxin A
  • a complementary strand of TALE repeat protein recognized single-strand DNA sequence and a complementary strand of mitochondrial ND4 gene sequence.
KeywordsDNA BINDING PROTEIN/DNA / TALE / DddA deaminase / DdCBE / dsDNA / ND4 / Mitochondrial base editor. / DNA BINDING PROTEIN-DNA complex
Function / homology
Function and homology information


Hydrolases; Acting on carbon-nitrogen bonds, other than peptide bonds; In cyclic amidines / toxin activity / hydrolase activity / metal ion binding / membrane
Similarity search - Function
Double-stranded DNA deaminase toxin A / Double-stranded DNA deaminase toxin A / Domain of unknown function DUF6531 / RHS protein / Domain of unknown function (DUF6531) / RHS protein / RHS repeat / RHS Repeat / PAAR motif / PAAR motif ...Double-stranded DNA deaminase toxin A / Double-stranded DNA deaminase toxin A / Domain of unknown function DUF6531 / RHS protein / Domain of unknown function (DUF6531) / RHS protein / RHS repeat / RHS Repeat / PAAR motif / PAAR motif / YD repeat / Rhs repeat-associated core / :
Similarity search - Domain/homology
DNA / DNA (> 10) / Double-stranded DNA deaminase toxin A
Similarity search - Component
Biological speciesXanthomonas (bacteria)
Burkholderia cenocepacia H111 (bacteria)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsJiangchao, X. / Wenchao, X. / Jia, C. / Bei, Y.
Funding support China, 3items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32070170 China
National Natural Science Foundation of China (NSFC)32371272 China
Ministry of Science and Technology (MoST, China)2023ZD0500501 China
CitationJournal: Mol Cell / Year: 2025
Title: Structural insights into DdCBE in action enable high-precision mitochondrial DNA editing.
Authors: Jiangchao Xiang / Wenchao Xu / Jing Wu / Yaxin Luo / Chengyu Liu / Yaofeng Hou / Jia Chen / Bei Yang /
Abstract: DddA-derived cytosine base editor (DdCBE) couples transcription activator-like effector (TALE) arrays and the double-stranded DNA (dsDNA)-specific cytidine deaminase DddA to target mitochondrial DNA ...DddA-derived cytosine base editor (DdCBE) couples transcription activator-like effector (TALE) arrays and the double-stranded DNA (dsDNA)-specific cytidine deaminase DddA to target mitochondrial DNA (mtDNA) for editing. However, structures of DdCBE in action are unavailable, impeding its mechanistic-based optimization for high-precision-demanding therapeutic applications. Here, we determined the cryo-electron microscopy (cryo-EM) structures of DdCBE targeting two native mitochondrial gene loci and combined editing data from systematically designed spacers to develop WinPred, a model that can predict DdCBE's editing outcome and guide its design to achieve high-precision editing. Furthermore, structure-guided engineering of DddA narrowed the editing window of DdCBE to 2-3 nt while minimizing its off-target (OT) editing to near-background levels, thereby generating accurate DdCBE (aDdCBE). Using aDdCBE, we precisely introduced a Leber hereditary optic neuropathy (LHON)-disease-related mutation into mtDNA and faithfully recapitulated the pathogenic conditions without interference from unintended bystander or OT mutations. Our work provides a mechanistic understanding of DdCBE and establishes WinPred and aDdCBE as useful tools for faithfully modeling or correcting disease-related mtDNA mutations.
History
DepositionSep 24, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Sep 17, 2025Provider: repository / Type: Initial release
Revision 1.1Sep 24, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update
Revision 1.2Oct 8, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: TALE repeat protein targeting mitochondiral ND1-L gene.
B: TALE repeat protein recognized single-strand DNA sequence and mitochondrial ND4 gene sequence.
C: a complementary strand of TALE repeat protein recognized single-strand DNA sequence and a complementary strand of mitochondrial ND4 gene sequence.
D: Double-stranded DNA deaminase toxin A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)104,8535
Polymers104,7884
Non-polymers651
Water181
1


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

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Components

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TALE repeat protein ... , 2 types, 2 molecules AB

#1: Protein TALE repeat protein targeting mitochondiral ND1-L gene.


Mass: 70525.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: TALE repeat protein targeting mitochondrial ND1-L gene sequence.
Source: (gene. exp.) Xanthomonas (bacteria) / Production host: Escherichia coli (E. coli)
#2: DNA chain TALE repeat protein recognized single-strand DNA sequence and mitochondrial ND4 gene sequence.


Mass: 10064.496 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: TALE repeat protein recognized single-strand DNA sequence and mitochondrial ND4 gene sequence.
Source: (synth.) Homo sapiens (human)

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DNA chain / Protein , 2 types, 2 molecules CD

#3: DNA chain a complementary strand of TALE repeat protein recognized single-strand DNA sequence and a complementary strand of mitochondrial ND4 gene sequence.


Mass: 10233.607 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: a complementary strand of TALE repeat protein recognized single-strand DNA sequence and a complementary strand of mitochondrial ND4 gene sequence.
Source: (synth.) Homo sapiens (human)
#4: Protein Double-stranded DNA deaminase toxin A / DddA / Cytidine deaminase / CD


Mass: 13964.678 Da / Num. of mol.: 1
Mutation: S1330I, A1341V, N1342S, E1370K, T1380I, T1413I, S1326G, G13348S, A1398V, S1418G
Source method: isolated from a genetically manipulated source
Details: DddA with additional mutations :DddA11 (including S1330I, A1341V, N1342S, E1370K, T1380I, and T1413I)and GSVG mutants (incluidng S1326G, G13348S, A1398V, and S1418G),and a Zn atom;
Source: (gene. exp.) Burkholderia cenocepacia H111 (bacteria)
Gene: dddA, I35_7839 / Production host: Escherichia coli (E. coli)
References: UniProt: P0DUH5, Hydrolases; Acting on carbon-nitrogen bonds, other than peptide bonds; In cyclic amidines

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Non-polymers , 2 types, 2 molecules

#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN
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: The complex of TALE protein-linked deaminase with a dsDNA substrate.
Type: COMPLEX / Details: TALE protein-DddA + dsDNA / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.106 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Burkholderia cenocepacia H111 (bacteria)1055524
31Xanthomonas (bacteria)338
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8 / Details: 20 mM Tris 8.0, 150 mM NaCl, 4 mM DTT
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMtrishydroxymethylaminomethaneTris1
2150 mMsodium chlorideNacl1
34 mMDithiothreitolDTT1
SpecimenConc.: 16 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K

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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 magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2953

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1696734
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 248533 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model building

3D fitting-ID: 1 / Chain-ID: A / Details: The initial model consisted of the D chain of current deposition complex / Pdb chain-ID: A / Source name: PDB / Type: experimental model

IDPDB-IDAccession codeChain residue rangeInitial refinement model-IDPdb chain residue range
18e5e

8e5e

8e5e1290-142711290-1427
23UGM

3ugm

3UGM1-6411-641
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0047257
ELECTRON MICROSCOPYf_angle_d0.58310166
ELECTRON MICROSCOPYf_dihedral_angle_d24.9041454
ELECTRON MICROSCOPYf_chiral_restr0.0391208
ELECTRON MICROSCOPYf_plane_restr0.0061104

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