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- PDB-8y9m: Cas12h1(D465A)-crRNA-dsDNA ternary complex -

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Basic information

Entry
Database: PDB / ID: 8y9m
TitleCas12h1(D465A)-crRNA-dsDNA ternary complex
Components
  • (DNA (29-mer)) x 2
  • Cas12h1 (D465A) mutant
  • crRNA
KeywordsIMMUNE SYSTEM/RNA/DNA / CRISPR-Cas / type V Cas effectors / IMMUNE SYSTEM / IMMUNE SYSTEM-RNA-DNA complex
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10)
Function and homology information
Biological speciesunidentified (others)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.76 Å
AuthorsZheng, W.W. / Liu, M.X.
Funding support China, 2items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)82225028 China
National Natural Science Foundation of China (NSFC)82172287 China
CitationJournal: Signal Transduct Target Ther / Year: 2025
Title: Molecular insights and rational engineering of a compact CRISPR-Cas effector Cas12h1 with a broad-spectrum PAM.
Authors: Weiwei Zheng / Hongyu Li / Mengxi Liu / Yuhang Wei / Bo Liu / Zekai Li / Chenyang Xiong / Shiqing Huang / Chunyi Hu / Songying Ouyang /
Abstract: Cas12h1 is a compact CRISPR-associated nuclease from functionally diverse type V CRISPR-Cas effectors and recognizes a purine-rich protospacer adjacent motif (PAM) distinct from that of other type V ...Cas12h1 is a compact CRISPR-associated nuclease from functionally diverse type V CRISPR-Cas effectors and recognizes a purine-rich protospacer adjacent motif (PAM) distinct from that of other type V Cas effectors. Here, we report the nickase preference of Cas12h1, which predominantly cleaves the nontarget strand (NTS) of a double-stranded DNA (dsDNA) substrate. In addition, Cas12h1 acts as a nickase in human cells. We further determined the cryo-EM structures of Cas12h1 in the surveillance, R-loop formation, and interference states, revealing the molecular mechanisms involved in the crRNA maturation, target recognition, R-loop formation, nuclease activation and target degradation. Cas12h1 notably recognizes a broad 5'-DHR-3' PAM (D is A, G, or T; H is A, C, or T; R is A or G) both in vitro and in human cells. In addition, Cas12h1 utilizes a distinct activation mechanism that the lid motif undergoes a "flexible to stable" transition to expose the catalytic site to the substrate. A high-fidelity nucleic acid detector, Cas12h1, was developed through rational engineering, which distinguishes single-base mismatches and retains comparable on-target activities. Our results shed light on the molecular mechanisms underlying Cas12h1 nickase, improve the understanding of type V Cas effectors, and expand the CRISPR toolbox for genome editing and molecular diagnosis.
History
DepositionFeb 7, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 12, 2025Provider: repository / Type: Initial release
Revision 1.1Mar 19, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cas12h1 (D465A) mutant
B: crRNA
C: DNA (29-mer)
D: DNA (29-mer)


Theoretical massNumber of molelcules
Total (without water)137,7604
Polymers137,7604
Non-polymers00
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 Cas12h1 (D465A) mutant


Mass: 99981.750 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 crRNA / RNA (62-mer)


Mass: 20041.965 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 (29-mer)


Mass: 8817.720 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (29-mer)


Mass: 8918.760 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
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: Ternary complex of Cas12h1 D465A mutant with crRNA and target DNA
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.1 MDa / Experimental value: YES
Source (natural)Organism: unidentified (others)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN 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: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.76 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 153309 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0049554
ELECTRON MICROSCOPYf_angle_d0.62213376
ELECTRON MICROSCOPYf_dihedral_angle_d18.3792117
ELECTRON MICROSCOPYf_chiral_restr0.041503
ELECTRON MICROSCOPYf_plane_restr0.0051325

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