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- PDB-8z0k: Cryo-EM structure of Cas8-HNH system at full R-loop state -

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Basic information

Entry
Database: PDB / ID: 8z0k
TitleCryo-EM structure of Cas8-HNH system at full R-loop state
Components
  • (type I-F CRISPR-associated ...) x 2
  • DNA (37-MER)
  • DNA (5'-D(P*GP*TP*GP*CP*GP*GP*A)-3')
  • HNH endonuclease
  • RNA (69-MER)
  • hypothetical protein J6N51_11000
KeywordsANTIVIRAL PROTEIN/DNA/RNA / a protein complex / ANTIVIRAL PROTEIN-DNA-RNA complex
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10)
Function and homology information
Biological speciesSelenomonas sp. (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.51 Å
AuthorsZhang, H. / Zhu, H. / Li, X. / Liu, Y.
Funding support China, 1items
OrganizationGrant numberCountry
Other government China
CitationJournal: EMBO J / Year: 2024
Title: Structural basis for the type I-F Cas8-HNH system.
Authors: Xuzichao Li / Yanan Liu / Jie Han / Lingling Zhang / Zhikun Liu / Lin Wang / Shuqin Zhang / Qian Zhang / Pengyu Fu / Hang Yin / Hongtao Zhu / Heng Zhang /
Abstract: The Cas3 nuclease is utilized by canonical type I CRISPR-Cas systems for processive target DNA degradation, while a newly identified type I-F CRISPR variant employs an HNH nuclease domain from the ...The Cas3 nuclease is utilized by canonical type I CRISPR-Cas systems for processive target DNA degradation, while a newly identified type I-F CRISPR variant employs an HNH nuclease domain from the natural fusion Cas8-HNH protein for precise target cleavage both in vitro and in human cells. Here, we report multiple cryo-electron microscopy structures of the type I-F Cas8-HNH system at different functional states. The Cas8-HNH Cascade complex adopts an overall G-shaped architecture, with the HNH domain occupying the C-terminal helical bundle domain (HB) of the Cas8 protein in canonical type I systems. The Linker region connecting Cas8-NTD and HNH domains adopts a rigid conformation and interacts with the Cas7.6 subunit, enabling the HNH domain to be in a functional position. The full R-loop formation displaces the HNH domain away from the Cas6 subunit, thus activating the target DNA cleavage. Importantly, our results demonstrate that precise target cleavage is dictated by a C-terminal helix of the HNH domain. Together, our work not only delineates the structural basis for target recognition and activation of the type I-F Cas8-HNH system, but also guides further developments leveraging this system for precise DNA editing.
History
DepositionApr 9, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Oct 2, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 9, 2024Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / em_entity_assembly / pdbx_entry_details / struct
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update / _em_admin.title / _em_entity_assembly.name / _struct.title
Revision 1.2Oct 30, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: type I-F CRISPR-associated protein Csy3
B: type I-F CRISPR-associated protein Csy3
C: type I-F CRISPR-associated protein Csy3
D: type I-F CRISPR-associated protein Csy3
E: hypothetical protein J6N51_11000
F: HNH endonuclease
G: type I-F CRISPR-associated protein Csy3
H: type I-F CRISPR-associated protein Csy3
I: DNA (37-MER)
J: DNA (5'-D(P*GP*TP*GP*CP*GP*GP*A)-3')
L: RNA (69-MER)
M: type I-F CRISPR-associated endoribonuclease Cas6/Csy4


Theoretical massNumber of molelcules
Total (without water)350,64012
Polymers350,64012
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Type I-F CRISPR-associated ... , 2 types, 7 molecules ABCDGHM

#1: Protein
type I-F CRISPR-associated protein Csy3


Mass: 37668.945 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Details: Molecular name from NCBI link:https://www.ncbi.nlm.nih.gov/protein/MBO6292766.1
Source: (gene. exp.) Selenomonas sp. (bacteria) / Production host: Escherichia coli (E. coli)
#7: Protein type I-F CRISPR-associated endoribonuclease Cas6/Csy4


Mass: 20735.873 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Molecular name from NCBI link:https://www.ncbi.nlm.nih.gov/protein/MBO6292765.1
Source: (gene. exp.) Selenomonas sp. (bacteria) / Production host: Escherichia coli (E. coli)

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Protein , 2 types, 2 molecules EF

#2: Protein hypothetical protein J6N51_11000


Mass: 28703.135 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Molecular name from NCBI link:https://www.ncbi.nlm.nih.gov/protein/MBO6292763.1
Source: (gene. exp.) Selenomonas sp. (bacteria) / Production host: Escherichia coli (E. coli)
#3: Protein HNH endonuclease


Mass: 39339.742 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Molecular name from NCBI link:https://www.ncbi.nlm.nih.gov/protein/MBO6292764.1
Source: (gene. exp.) Selenomonas sp. (bacteria) / Production host: Escherichia coli (E. coli)

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DNA chain , 2 types, 2 molecules IJ

#4: DNA chain DNA (37-MER)


Mass: 11271.250 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Selenomonas sp. (bacteria) / Production host: Escherichia coli (E. coli)
#5: DNA chain DNA (5'-D(P*GP*TP*GP*CP*GP*GP*A)-3')


Mass: 2178.447 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Selenomonas sp. (bacteria) / Production host: Escherichia coli (E. coli)

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RNA chain , 1 types, 1 molecules L

#6: RNA chain RNA (69-MER)


Mass: 22398.293 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Selenomonas sp. (bacteria) / Production host: Escherichia coli (E. coli)

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Details

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: Cryo-EM structure of Cas8-HNH system at full R-loop state
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Selenomonas sp. (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

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1600 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 2.51 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 173047 / Symmetry type: POINT

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