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- EMDB-61646: Cryo-EM structure of the mono-DdCBE bound to a dsDNA substrate. -

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Basic information

Entry
Database: EMDB / ID: EMD-61646
TitleCryo-EM structure of the mono-DdCBE bound to a dsDNA substrate.
Map dataPrimary map of TALE protein - deaminase bound in dsDNA substrate complex
Sample
  • Complex: The complex of TALE protein-linked deaminase with a dsDNA substrate.
    • Protein or peptide: TALE repeat protein targeting mitochondiral ND1-L gene.
    • DNA: TALE repeat protein recognized single-strand DNA sequence and mitochondrial ND4 gene sequence.
    • DNA: a complementary strand of TALE repeat protein recognized single-strand DNA sequence and a complementary strand of mitochondrial ND4 gene sequence.
    • Protein or peptide: Double-stranded DNA deaminase toxin A
  • Ligand: ZINC ION
  • Ligand: water
KeywordsTALE / DddA deaminase / DdCBE / dsDNA / ND4 / Mitochondrial base editor. / DNA BINDING PROTEIN/DNA / 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
Double-stranded DNA deaminase toxin A
Similarity search - Component
Biological speciesBurkholderia cenocepacia H111 (bacteria) / Xanthomonas (bacteria) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsJiangchao X / Wenchao X / Jia C / Bei Y
Funding support China, 3 items
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, 2024-
Header (metadata) releaseSep 17, 2025-
Map releaseSep 17, 2025-
UpdateOct 8, 2025-
Current statusOct 8, 2025Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_61646.png

Downloads & links

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Map

FileDownload / File: emd_61646.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPrimary map of TALE protein - deaminase bound in dsDNA substrate complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.96 Å/pix.
x 360 pix.
= 347.148 Å		0.96 Å/pix.
x 360 pix.
= 347.148 Å		0.96 Å/pix.
x 360 pix.
= 347.148 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.9643 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0943
Minimum - Maximum-0.0017671458 - 1.9882239
Average (Standard dev.)0.0003723802 (±0.014423497)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 347.14798 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_61646_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half 2 map of TALE protein - deaminase...

Fileemd_61646_half_map_1.map
AnnotationHalf 2 map of TALE protein - deaminase bound in dsDNA substrate complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half 1 map of TALE protein - deaminase...

Fileemd_61646_half_map_2.map
AnnotationHalf 1 map of TALE protein - deaminase bound in dsDNA substrate complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : The complex of TALE protein-linked deaminase with a dsDNA substrate.

EntireName: The complex of TALE protein-linked deaminase with a dsDNA substrate.
Components
  • Complex: The complex of TALE protein-linked deaminase with a dsDNA substrate.
    • Protein or peptide: TALE repeat protein targeting mitochondiral ND1-L gene.
    • DNA: TALE repeat protein recognized single-strand DNA sequence and mitochondrial ND4 gene sequence.
    • DNA: a complementary strand of TALE repeat protein recognized single-strand DNA sequence and a complementary strand of mitochondrial ND4 gene sequence.
    • Protein or peptide: Double-stranded DNA deaminase toxin A
  • Ligand: ZINC ION
  • Ligand: water

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Supramolecule #1: The complex of TALE protein-linked deaminase with a dsDNA substrate.

SupramoleculeName: The complex of TALE protein-linked deaminase with a dsDNA substrate.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 / Details: TALE protein-DddA + dsDNA
Source (natural)Organism: Burkholderia cenocepacia H111 (bacteria)
Molecular weightTheoretical: 106 KDa

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

MacromoleculeName: TALE repeat protein targeting mitochondiral ND1-L gene.
type: protein_or_peptide / ID: 1
Details: TALE repeat protein targeting mitochondrial ND1-L gene sequence.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Xanthomonas (bacteria)
Molecular weightTheoretical: 70.525031 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSDIADLR TLGYSQQQQE KIKPKVRSTV AQHHEALVGH GFTHAHIVAL SQHPAALGTV AVKYQDMIAA LPEATHEAIV GVGKQWSGA RALEALLTVA GELRGPPLQL DTGQLLKIAK RGGVTAVEAV HAWRNALTGA PLNLTPEQVV AIASHDGGKQ A LETVQRLL ...String:
MGSSDIADLR TLGYSQQQQE KIKPKVRSTV AQHHEALVGH GFTHAHIVAL SQHPAALGTV AVKYQDMIAA LPEATHEAIV GVGKQWSGA RALEALLTVA GELRGPPLQL DTGQLLKIAK RGGVTAVEAV HAWRNALTGA PLNLTPEQVV AIASHDGGKQ A LETVQRLL PVLCQAHGLT PEQVVAIASN GGGKQALETV QRLLPVLCQA HGLTPEQVVA IASNIGGKQA LETVQRLLPV LC QAHGLTP EQVVAIASNN GGKQALETVQ RLLPVLCQAH GLTPEQVVAI ASHDGGKQAL ETVQRLLPVL CQAHGLTPEQ VVA IASHDG GKQALETVQR LLPVLCQAHG LTPEQVVAIA SNGGGKQALE TVQRLLPVLC QAHGLTPEQV VAIASNIGGK QALE TVQRL LPVLCQAHGL TPEQVVAIAS NNGGKQALET VQRLLPVLCQ AHGLTPEQVV AIASHDGGKQ ALETVQRLLP VLCQA HGLT PEQVVAIASH DGGKQALETV QRLLPVLCQA HGLTPEQVVA IASNNGGKQA LETVQRLLPV LCQAHGLTPE QVVAIA SNG GGKQALETVQ RLLPVLCQAH GLTPEQVVAI ASNGGGKQAL ETVQRLLPVL CQAHGLTPEQ VVAIASNGGG KQALESI VA QLSRPDPALA ALTNDHLVAL ACLGGRPALD AVKKGLG

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Macromolecule #4: Double-stranded DNA deaminase toxin A

MacromoleculeName: Double-stranded DNA deaminase toxin A / type: protein_or_peptide / ID: 4
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;
Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on carbon-nitrogen bonds, other than peptide bonds; In cyclic amidines
Source (natural)Organism: Burkholderia cenocepacia H111 (bacteria)
Molecular weightTheoretical: 13.964678 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSYALGPYQI SAPQLPAYNG QTVGTFYYVN DAGGLEGKVF ISGGPTPYPN YVSAGHVESQ SALFMRDNGI SEGLVFHNNP KGTCGFCVN MIETLLPENA KMTVVPPEGV IPVKRGATGE TKVFIGNSNG PKSP

UniProtKB: Double-stranded DNA deaminase toxin A

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Macromolecule #2: TALE repeat protein recognized single-strand DNA sequence and mit...

MacromoleculeName: TALE repeat protein recognized single-strand DNA sequence and mitochondrial ND4 gene sequence.
type: dna / ID: 2
Details: TALE repeat protein recognized single-strand DNA sequence and mitochondrial ND4 gene sequence.
Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 10.064496 KDa
SequenceString:
(DG)(DA)(DA)(DT)(DC)(DT)(DA)(DG)(DC)(DC) (DT)(DA)(DG)(DC)(DC)(DG)(DT)(DT)(DT)(DC) (DC)(DT)(DG)(DA)(DT)(DC)(DA)(DA)(DA) (DT)(DA)(DT)(DC)

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Macromolecule #3: a complementary strand of TALE repeat protein recognized single-s...

MacromoleculeName: a complementary strand of TALE repeat protein recognized single-strand DNA sequence and a complementary strand of mitochondrial ND4 gene sequence.
type: dna / ID: 3
Details: a complementary strand of TALE repeat protein recognized single-strand DNA sequence and a complementary strand of mitochondrial ND4 gene sequence.
Number of copies: 1 / Classification: DNA
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 10.233607 KDa
SequenceString:
(DG)(DA)(DT)(DA)(DT)(DT)(DT)(DG)(DA)(DT) (DC)(DA)(DG)(DG)(DA)(DA)(DA)(DC)(DG)(DG) (DC)(DT)(DA)(DG)(DG)(DC)(DT)(DA)(DG) (DA)(DT)(DT)(DC)

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 1 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration16 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMTristrishydroxymethylaminomethane
150.0 mMNaclsodium chloride
4.0 mMDTTDithiothreitol

Details: 20 mM Tris 8.0, 150 mM NaCl, 4 mM DTT
GridModel: Quantifoil R0.6/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 2953 / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1696734
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3ugm

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 248533
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

Initial model
PDB IDChainDetails

8e5e

chain_id: A, residue_range: 1290-1427, source_name: PDB, initial_model_type: experimental modelThe initial model consisted of the D chain of current deposition complex

3ugm

chain_id: A, residue_range: 1-641, source_name: PDB, initial_model_type: experimental modelThe initial model consisted of the D chain of current deposition complex
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

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

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