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- PDB-9jg3: Structure of Cas12p-TrxA-guide RNA-target DNA complex(33-bp dsDNA) -

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Basic information

Entry
Database: PDB / ID: 9jg3
TitleStructure of Cas12p-TrxA-guide RNA-target DNA complex(33-bp dsDNA)
Components
  • (DNA (33-MER)) x 2
  • Cas12p
  • RNA (246-MER)
  • Thioredoxin
KeywordsRNA BINDING PROTEIN/RNA/DNA / CRISPR-Cas / nuclease / Heterodimer / RNA BINDING PROTEIN-RNA-DNA complex
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / :
Function and homology information
Biological speciesunidentified (others)
Escherichia coli BL21 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsWang, Z.P. / Wang, Y.J. / Ji, Q.J.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)22277078 China
CitationJournal: Nat Microbiol / Year: 2026
Title: Phage-associated Cas12p nucleases require binding to bacterial thioredoxin for activation and cleavage of target DNA.
Authors: Zhipeng Wang / Yujue Wang / Hui Gao / Jiani Dai / Na Tang / Yannan Wang / Quanjiang Ji /
Abstract: The evolutionary competition within phage-host systems led to the emergence of CRISPR-Cas defence mechanisms in bacteria and anti-CRISPR elements in bacteriophages. Although anti-CRISPR elements are ...The evolutionary competition within phage-host systems led to the emergence of CRISPR-Cas defence mechanisms in bacteria and anti-CRISPR elements in bacteriophages. Although anti-CRISPR elements are well characterized, the role of bacterial factors that influence CRISPR-Cas efficacy has been comparatively overlooked. Type V CRISPR-Cas12 systems display striking functional and mechanistic diversity for nucleic acid targeting. Here we use a bioinformatic approach to identify Cas12p, a phage-associated nuclease that forms complexes with the bacterial thioredoxin protein TrxA to enable target DNA degradation. This represents an unexpected phage-bacteria interaction, in which the bacteriophage co-opts a bacterial factor to augment its own genome degradation machinery, potentially against competing phages. Biochemical characterization, cryo-EM-based structural analysis of the Cas12p-TrxA-sgRNA-dsDNA complex at 2.67 Å and bacterial defence assays reveal that TrxA directly binds and activates Cas12p, enabling its nuclease activity and subsequent CRISPR immunity. These findings expand our understanding of the multilayered intricacies of phage-bacteria molecular interactions.
History
DepositionSep 5, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Sep 10, 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: Cas12p
B: RNA (246-MER)
C: DNA (33-MER)
D: DNA (33-MER)
E: Thioredoxin


Theoretical massNumber of molelcules
Total (without water)196,2745
Polymers196,2745
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Cas12p


Mass: 84964.945 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) unidentified (others) / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: RNA chain RNA (246-MER)


Mass: 79190.891 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) unidentified (others) / Production host: Escherichia coli BL21(DE3) (bacteria)
#3: DNA chain DNA (33-MER)


Mass: 10325.686 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (33-MER)


Mass: 9973.404 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: Protein Thioredoxin


Mass: 11818.582 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A140SSC0
Has protein modificationY

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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: complex of Cas12p-TrxA-guide RNA-target DNA (33-bp dsDNA)
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Source (natural)Organism: unidentified (others)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenConc.: 3.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Homemade
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: 1200 nm
Image recordingElectron dose: 20 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.21.1_5286:model refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 66342 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0059737
ELECTRON MICROSCOPYf_angle_d0.59713972
ELECTRON MICROSCOPYf_dihedral_angle_d21.1723127
ELECTRON MICROSCOPYf_chiral_restr0.0411667
ELECTRON MICROSCOPYf_plane_restr0.0051142

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