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- PDB-9irf: Cryo-EM Structure of csy1-4 with crRNA -

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Basic information

Entry
Database: PDB / ID: 9irf
TitleCryo-EM Structure of csy1-4 with crRNA
Components
  • CRISPR-associated endonuclease Cas6f/Csy4
  • CRISPR-associated protein Csy1
  • CRISPR-associated protein Csy2
  • CRISPR-associated protein Csy3
  • crRNA (87-MER)
KeywordsRNA BINDING PROTEIN/RNA / crRNA / cas6f / cas7f / cas5f / cas8f / 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 / RNA binding
Similarity search - Function
CRISPR-associated protein Csy1 / CRISPR-associated protein (Cas_Csy1) / CRISPR-associated endoribonuclease Cas6/Csy4, subtype I-F/YPEST / CRISPR-associated endoribonuclease Cas6/Csy4, subtype I-F/YPEST superfamily / CRISPR-associated protein (Cas_Csy4) / CRISPR-associated protein Csy2 / CRISPR-associated protein (Cas_Csy2) / CRISPR-associated protein Csy3 / CRISPR-associated protein (Cas_Csy3)
Similarity search - Domain/homology
: / RNA / RNA (> 10) / CRISPR-associated endonuclease Cas6f/Csy4 / CRISPR-associated protein Csy3 / CRISPR-associated protein Csy2 / CRISPR-associated protein Csy1
Similarity search - Component
Biological speciesPectobacterium atrosepticum SCRI1043 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsGao, X. / Cui, S. / Zhu, H. / Zhu, K. / Shang, K.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Mol Cell / Year: 2025
Title: RNA anti-CRISPRs deplete Cas proteins to inhibit the CRISPR-Cas system.
Authors: Xiaopan Gao / Kaixiang Zhu / Weihe Zhang / Lin Wang / Linyue Wang / Lei Hua / Tongxin Niu / Bo Qin / Xia Yu / Hongtao Zhu / Sheng Cui /
Abstract: RNA-based anti-CRISPRs (Racrs) interfere with the type I-F CRISPR-Cas system by mimicking the repeats found in CRISPR arrays. Here, we determined the cryo-electron microscopy (cryo-EM) structures of ...RNA-based anti-CRISPRs (Racrs) interfere with the type I-F CRISPR-Cas system by mimicking the repeats found in CRISPR arrays. Here, we determined the cryo-electron microscopy (cryo-EM) structures of the type I-F crRNA-guided surveillance complex (Csy complex) from Pectobacterium atrosepticum and three RacrIF1-induced aberrant subcomplexes. Additionally, we observed that Cas7f proteins could bind to non-specific nucleic acids, forming right-handed superhelical filaments composed of different Cas7 copies. Mechanistically, RacrIF1 lacks the specific S-conformation observed in the corresponding position of the 5' handle in canonical CRISPR complexes, and it instead adopts a periodic "5 + 1" pattern. This conformation creates severe steric hindrance for Cas5f-Cas8f heterodimer and undermines their binding. Furthermore, Cas7f nonspecifically binds nucleic acids and can form infinite superhelical filaments along Racrs molecules. This oligomerization sequesters Cas6f and Cas7f from binding, therefore blocking the formation of functional CRISPR-Cas effector complexes and ultimately blocking antiviral immunity. Our study provides a structural basis underlying Racrs-mediated CRISPRs inhibition.
History
DepositionJul 15, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Jan 14, 2026Provider: repository / Type: Initial release
Revision 1.0Jan 14, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jan 14, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jan 14, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jan 14, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jan 14, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jan 14, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CRISPR-associated protein Csy2
B: CRISPR-associated endonuclease Cas6f/Csy4
C: CRISPR-associated protein Csy3
D: CRISPR-associated protein Csy3
E: CRISPR-associated protein Csy3
F: CRISPR-associated protein Csy3
G: CRISPR-associated protein Csy3
H: CRISPR-associated protein Csy3
I: crRNA (87-MER)
J: CRISPR-associated protein Csy1


Theoretical massNumber of molelcules
Total (without water)355,59610
Polymers355,59610
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 CRISPR-associated protein Csy2


Mass: 34903.383 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pectobacterium atrosepticum SCRI1043 (bacteria)
Gene: csy2, ECA3682 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6D0W7
#2: Protein CRISPR-associated endonuclease Cas6f/Csy4 / crRNA endonuclease


Mass: 20487.529 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pectobacterium atrosepticum SCRI1043 (bacteria)
Gene: cas6f, csy4, ECA3684 / Production host: Escherichia coli (E. coli)
References: UniProt: Q6D0W5, Hydrolases; Acting on ester bonds
#3: Protein
CRISPR-associated protein Csy3


Mass: 36948.512 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pectobacterium atrosepticum SCRI1043 (bacteria)
Gene: csy3, ECA3683 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6D0W6
#4: RNA chain crRNA (87-MER)


Mass: 28060.721 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pectobacterium atrosepticum SCRI1043 (bacteria)
Production host: Escherichia coli (E. coli) / References: GenBank: 49609491
#5: Protein CRISPR-associated protein Csy1


Mass: 50453.484 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pectobacterium atrosepticum SCRI1043 (bacteria)
Gene: csy1, ECA3681 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6D0W8
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: csy1-4 complex of CRISPR with crRNA / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Pectobacterium atrosepticum SCRI1043 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil R1.2/1.3
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 magnification: 2500 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1800 nm / Calibrated defocus max: 2500 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 55 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 71806 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00523020
ELECTRON MICROSCOPYf_angle_d0.76631500
ELECTRON MICROSCOPYf_dihedral_angle_d10.0913672
ELECTRON MICROSCOPYf_chiral_restr0.0443509
ELECTRON MICROSCOPYf_plane_restr0.0063912

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