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- PDB-8yb6: Type I-EHNH Cascade complex -

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Basic information

Entry
Database: PDB / ID: 8yb6
TitleType I-EHNH Cascade complex
Components
  • (CRISPR system Cascade subunit ...) x 2
  • (CRISPR-associated protein ...) x 2
  • 61-nt crRNA
  • CRISPR-associated endoribonuclease Cse3
KeywordsRNA BINDING PROTEIN/RNA / RNA pro / RNA BINDING PROTEIN-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: 3.06 Å
AuthorsLi, Z.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Mol Cell / Year: 2024
Title: Mechanisms for HNH-mediated target DNA cleavage in type I CRISPR-Cas systems.
Authors: Chendi Zhang / Fugen Chen / Feng Wang / Haijiang Xu / Jialin Xue / Zhuang Li /
Abstract: The metagenome-derived type I-E and type I-F variant CRISPR-associated complex for antiviral defense (Cascade) complexes, fused with HNH domains, precisely cleave target DNA, representing recently ...The metagenome-derived type I-E and type I-F variant CRISPR-associated complex for antiviral defense (Cascade) complexes, fused with HNH domains, precisely cleave target DNA, representing recently identified genome editing tools. However, the underlying working mechanisms remain unknown. Here, structures of type I-F and I-E Cascade complexes at different states are reported. In type I-F Cascade, Cas8f loosely attaches to Cascade head and is adjacent to the 5' end of the target single-stranded DNA (ssDNA). Formation of the full R-loop drives the Cascade head to move outward, allowing Cas8f to detach and rotate ∼150° to accommodate target ssDNA for cleavage. In type I-E Cascade, Cas5e domain is adjacent to the 5' end of the target ssDNA. Full crRNA-target pairing drives the lift of the Cascade head, widening the substrate channel for target ssDNA entrance. Altogether, these analyses into both complexes revealed that crRNA-guided positioning of target DNA and target DNA-induced HNH unlocking are two key factors for their site-specific cleavage of target DNA.
History
DepositionFeb 11, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Jul 31, 2024Provider: repository / Type: Initial release
Revision 1.1Aug 7, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update
Revision 1.2Sep 11, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR system Cascade subunit CasD
B: CRISPR-associated endoribonuclease Cse3
C: 61-nt crRNA
D: CRISPR system Cascade subunit CasC
E: CRISPR system Cascade subunit CasC
F: CRISPR system Cascade subunit CasC
G: CRISPR system Cascade subunit CasC
H: CRISPR system Cascade subunit CasC
I: CRISPR system Cascade subunit CasC
J: CRISPR-associated protein Cse1 (CRISPR_cse1)
K: CRISPR-associated protein Cse2 (CRISPR_cse2)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)426,38513
Polymers426,25411
Non-polymers1312
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 system Cascade subunit ... , 2 types, 7 molecules ADEFGHI

#1: Protein CRISPR system Cascade subunit CasD


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 BL21(DE3) (bacteria) / References: UniProt: A0A1V6F8C5
#4: Protein
CRISPR system Cascade subunit CasC


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 BL21(DE3) (bacteria) / References: UniProt: A0A1V6F8B5

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CRISPR-associated protein ... , 2 types, 2 molecules JK

#5: 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 BL21(DE3) (bacteria) / References: UniProt: A0A1V6F8D1
#6: Protein CRISPR-associated protein Cse2 (CRISPR_cse2)


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 BL21(DE3) (bacteria) / References: UniProt: A0A1V6F8C9

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

#2: Protein CRISPR-associated endoribonuclease Cse3


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 BL21(DE3) (bacteria)
References: UniProt: A0A1V6F8C4, Hydrolases; Acting on ester bonds
#3: RNA chain 61-nt crRNA


Mass: 19681.744 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 BL21(DE3) (bacteria)
#7: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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: Type I-EHNH Cascade complex / Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Source (natural)Organism: Candidatus Cloacimonetes bacterium ADurb.Bin088 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 6.2
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: 2200 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 54 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 291872 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00528509
ELECTRON MICROSCOPYf_angle_d0.79538897
ELECTRON MICROSCOPYf_dihedral_angle_d9.8574361
ELECTRON MICROSCOPYf_chiral_restr0.0444283
ELECTRON MICROSCOPYf_plane_restr0.0054831

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