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- PDB-8yeo: Type I-FHNH Cascade-dsDNA R-loop complex -

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Basic information

Entry
Database: PDB / ID: 8yeo
TitleType I-FHNH Cascade-dsDNA R-loop complex
Components
  • 60-nt crRNA
  • Cas5f
  • Cas6f
  • Cas7f
  • Cas8f fusion with HNH
  • NTS
  • TS
KeywordsRNA BINDING PROTEIN/DNA/RNA / protein / RNA / RNA BINDING PROTEIN-DNA-RNA complex
Function / homologyDNA/RNA hybrid / DNA/RNA hybrid (> 10) / RNA / RNA (> 10)
Function and homology information
Biological speciesSelenomonas sp. (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.44 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 22, 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: Cas5f
J: Cas8f fusion with HNH
B: Cas6f
I: Cas7f
C: 60-nt crRNA
T: TS
D: Cas7f
E: Cas7f
F: Cas7f
G: Cas7f
H: Cas7f
N: NTS


Theoretical massNumber of molelcules
Total (without water)359,03012
Polymers359,03012
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

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Protein , 4 types, 9 molecules AJBIDEFGH

#1: Protein Cas5f


Mass: 28703.135 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Selenomonas sp. (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Protein Cas8f fusion with HNH


Mass: 39339.742 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Selenomonas sp. (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)
#3: Protein Cas6f


Mass: 20735.873 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Selenomonas sp. (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)
#4: Protein
Cas7f


Mass: 38700.172 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Selenomonas sp. (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)

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RNA chain , 1 types, 1 molecules C

#5: RNA chain 60-nt crRNA


Mass: 19440.611 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Selenomonas sp. (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)

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DNA/RNA hybrid , 2 types, 2 molecules TN

#6: DNA/RNA hybrid TS


Mass: 15068.426 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Selenomonas sp. (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)
#7: DNA/RNA hybrid NTS


Mass: 3541.243 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Selenomonas sp. (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)

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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-FHNH Cascade-dsDNA R-loop complex / Type: COMPLEX / Entity ID: #1-#3, #5-#6, #4, #7 / Source: RECOMBINANT
Source (natural)Organism: Selenomonas sp. (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7
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: 3.44 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 48756 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00324973
ELECTRON MICROSCOPYf_angle_d0.60534258
ELECTRON MICROSCOPYf_dihedral_angle_d15.8864222
ELECTRON MICROSCOPYf_chiral_restr0.0413797
ELECTRON MICROSCOPYf_plane_restr0.0043969

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