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- PDB-8zm3: Cryo-EM strcuture of Cas5-HNH Cascade,apo-Conf2 -

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Basic information

Entry
Database: PDB / ID: 8zm3
TitleCryo-EM strcuture of Cas5-HNH Cascade,apo-Conf2
Components
  • (CRISPR system Cascade subunit ...) x 2
  • (CRISPR-associated protein ...) x 2
  • CRISPR-associated endoribonuclease Cse3
  • RNA (61-MER)
KeywordsIMMUNE SYSTEM/RNA / Cryo-EM strcuture / Cascade / CRISPR-Cas / IMMUNE SYSTEM-RNA complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / hydrolase activity / RNA binding / zinc ion binding
Similarity search - Function
CRISPR-associated protein, CT1975 / CRISPR-associated protein Cse2 / Cse2 superfamily / CT1975-like protein / CRISPR-associated protein Cse2 (CRISPR_cse2) / CRISPR-associated protein, CasD / CRISPR-associated protein Cse1 / CRISPR-associated protein Cse1 (CRISPR_cse1) / CRISPR-associated protein Cse3 / CRISPR associated protein ...CRISPR-associated protein, CT1975 / CRISPR-associated protein Cse2 / Cse2 superfamily / CT1975-like protein / CRISPR-associated protein Cse2 (CRISPR_cse2) / CRISPR-associated protein, CasD / CRISPR-associated protein Cse1 / CRISPR-associated protein Cse1 (CRISPR_cse1) / CRISPR-associated protein Cse3 / CRISPR associated protein / CRISPR_assoc / HNH endonuclease / HNH endonuclease / CRISPR-associated protein, Cas5 / CRISPR-associated protein (Cas_Cas5) / CRISPR-associated protein Cas5, N-terminal / HNH nucleases / HNH nuclease
Similarity search - Domain/homology
RNA / RNA (> 10) / CRISPR system Cascade subunit CasC / CRISPR-associated endoribonuclease Cse3 / CRISPR system Cascade subunit CasD / CRISPR-associated protein Cse2 (CRISPR_cse2) / CRISPR-associated protein Cse1 (CRISPR_cse1)
Similarity search - Component
Biological speciesCandidatus Cloacimonetes bacterium ADurb.Bin088 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsLiu, Y.N. / Wang, L. / Zhang, H. / Zhu, H.
Funding support China, 1items
OrganizationGrant numberCountry
Chinese Academy of Sciences China
CitationJournal: Nat Commun / Year: 2025
Title: Structural basis for RNA-guided DNA degradation by Cas5-HNH/Cascade complex.
Authors: Yanan Liu / Lin Wang / Qian Zhang / Pengyu Fu / Lingling Zhang / Ying Yu / Heng Zhang / Hongtao Zhu /
Abstract: Type I-E CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated proteins) system is one of the most extensively studied RNA-guided adaptive immune systems in ...Type I-E CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated proteins) system is one of the most extensively studied RNA-guided adaptive immune systems in prokaryotes, providing defense against foreign genetic elements. Unlike the previously characterized Cas3 nuclease, which exhibits progressive DNA cleavage in the typical type I-E system, a recently identified HNH-comprising Cascade system enables precise DNA cleavage. Here, we present several near-atomic cryo-electron microscopy (cryo-EM) structures of the Candidatus Cloacimonetes bacterium Cas5-HNH/Cascade complex, both in its DNA-bound and unbound states. Our analysis reveals extensive interactions between the HNH domain and adjacent subunits, including Cas6 and Cas11, with mutations in these key interactions significantly impairing enzymatic activity. Upon DNA binding, the Cas5-HNH/Cascade complex adopts a more compact conformation, with subunits converging toward the center of nuclease, leading to its activation. Notably, we also find that divalent ions such as zinc, cobalt, and nickel down-regulate enzyme activity by destabilizing the Cascade complex. Together, these findings offer structural insights into the assembly and activation of the Cas5-HNH/Cascade complex.
History
DepositionMay 22, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Dec 11, 2024Provider: repository / Type: Initial release
Revision 1.1Jun 18, 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: RNA (61-MER)
C: CRISPR-associated protein Cse1 (CRISPR_cse1)
E: CRISPR-associated protein Cse2 (CRISPR_cse2)
F: CRISPR system Cascade subunit CasC
H: CRISPR system Cascade subunit CasC
I: CRISPR system Cascade subunit CasC
J: CRISPR system Cascade subunit CasC
K: CRISPR system Cascade subunit CasC
G: CRISPR system Cascade subunit CasC
B: CRISPR system Cascade subunit CasD
D: CRISPR-associated endoribonuclease Cse3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)426,31913
Polymers426,27011
Non-polymers492
Water00
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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CRISPR-associated protein ... , 2 types, 2 molecules CE

#2: Protein CRISPR-associated protein Cse1 (CRISPR_cse1)


Mass: 60665.359 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Candidatus Cloacimonetes bacterium ADurb.Bin088 (bacteria)
Gene: BWX75_00747 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A1V6F8D1
#3: Protein CRISPR-associated protein Cse2 (CRISPR_cse2) / Cas11


Mass: 20300.639 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Candidatus Cloacimonetes bacterium ADurb.Bin088 (bacteria)
Gene: BWX75_00748 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A1V6F8C9

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CRISPR system Cascade subunit ... , 2 types, 7 molecules FHIJKGB

#4: Protein
CRISPR system Cascade subunit CasC / Cas7


Mass: 41794.367 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Candidatus Cloacimonetes bacterium ADurb.Bin088 (bacteria)
Gene: casC, BWX75_00749 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A1V6F8B5
#5: Protein CRISPR system Cascade subunit CasD / Cas5


Mass: 43621.383 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Candidatus Cloacimonetes bacterium ADurb.Bin088 (bacteria)
Gene: casD, BWX75_00750 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A1V6F8C5

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RNA chain / Protein / Non-polymers , 3 types, 4 molecules AD

#1: RNA chain RNA (61-MER)


Mass: 19697.695 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Candidatus Cloacimonetes bacterium ADurb.Bin088 (bacteria)
Production host: Escherichia coli (E. coli)
#6: Protein CRISPR-associated endoribonuclease Cse3 / Cas6


Mass: 31218.768 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Candidatus Cloacimonetes bacterium ADurb.Bin088 (bacteria)
Gene: cse3, BWX75_00751 / Production host: Escherichia coli (E. coli)
References: UniProt: A0A1V6F8C4, Hydrolases; Acting on ester bonds
#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg

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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: Cas5-HNH Cascade / Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Molecular weightValue: 400 kDa/nm / Experimental value: YES
Source (natural)Organism: Candidatus Cloacimonetes bacterium ADurb.Bin088 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 25000 nm / Nominal defocus min: 12000 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 20320 / Symmetry type: POINT
RefinementHighest resolution: 3.1 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01127663
ELECTRON MICROSCOPYf_angle_d1.24237800
ELECTRON MICROSCOPYf_dihedral_angle_d10.834263
ELECTRON MICROSCOPYf_chiral_restr0.054216
ELECTRON MICROSCOPYf_plane_restr0.0084695

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