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- PDB-9ky4: Cryo-EM structure of the mono-DdCBE bound TS substrate complex. -

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Basic information

Entry
Database: PDB / ID: 9ky4
TitleCryo-EM structure of the mono-DdCBE bound TS substrate complex.
Components
  • A complementary strand of TALE repeat protein recognized single-strand DNA sequence and mitochondrial ND5.1 gene sequence.
  • Double-stranded DNA deaminase toxin A
  • TALE repeat protein
  • TALE repeat protein recognized single-strand DNA sequence and mitochondrial ND5.1 gene sequence.
KeywordsDNA BINDING PROTEIN/DNA / TALE / cytosine 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. / 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
DepositionDec 8, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Oct 1, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: TALE repeat protein
B: TALE repeat protein recognized single-strand DNA sequence and mitochondrial ND5.1 gene sequence.
C: A complementary strand of TALE repeat protein recognized single-strand DNA sequence and mitochondrial ND5.1 gene sequence.
D: Double-stranded DNA deaminase toxin A
hetero molecules


Theoretical massNumber of molelcules
Total (without water)106,1365
Polymers106,0714
Non-polymers651
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 TALE repeat protein


Mass: 70525.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: The TALE repeat protein recognized 16 bp-length mitochondrial ND1 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 ND5.1 gene sequence.


Mass: 10649.845 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#3: DNA chain A complementary strand of TALE repeat protein recognized single-strand DNA sequence and mitochondrial ND5.1 gene sequence.


Mass: 10886.021 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#4: Protein Double-stranded DNA deaminase toxin A / DddA / Cytidine deaminase / CD


Mass: 14009.717 Da / Num. of mol.: 1
Mutation: S1330I, A1341V, N1342S, E1347A, E1370K, T1380I, T1413I
Source method: isolated from a genetically manipulated source
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
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
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 an ND51-dsDNA substrate.
Type: COMPLEX / Details: TALE protein-DddA + dsDNA / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.110 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Burkholderia cenocepacia (bacteria)95486
21Xanthomonas (bacteria)338
31Homo sapiens (human)9606
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: 2340

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARCv4particle selectiontemplate picking and topaz picking
2PHENIX1.20.1_4487:model refinement
13cryoSPARCv43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3319584
Details: initial particles from the template picker and Topaz picking
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 343135 / 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

3UGM1-64121-641
RefinementHighest resolution: 3 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0037260
ELECTRON MICROSCOPYf_angle_d0.63110171
ELECTRON MICROSCOPYf_dihedral_angle_d22.0642749
ELECTRON MICROSCOPYf_chiral_restr0.0391210
ELECTRON MICROSCOPYf_plane_restr0.0051104

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