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- PDB-7wwu: ICP1 Csy complex -

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Basic information

Entry
Database: PDB / ID: 7wwu
TitleICP1 Csy complex
Components
  • Csy1
  • Csy2
  • Csy3
  • Csy4
  • guide-RNA
KeywordsRNA BINDING PROTEIN/RNA / CRISPR-Cas system / RNA / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


maintenance of CRISPR repeat elements / endonuclease activity
Similarity search - Function
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) / Csy3 / Csy2 / Csy1 / Csy4
Similarity search - Component
Biological speciesVibrio phage ICP1_2011_A (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsZhang, M. / Peng, R.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
Citation
Journal: Proc Natl Acad Sci U S A / Year: 2023
Title: Mechanistic insights into DNA binding and cleavage by a compact type I-F CRISPR-Cas system in bacteriophage.
Authors: Manling Zhang / Ruchao Peng / Qi Peng / Sheng Liu / Zhiteng Li / Yuqin Zhang / Hao Song / Jia Yang / Xiao Xing / Peiyi Wang / Jianxun Qi / George F Gao /
Abstract: CRISPR-Cas systems are widespread adaptive antiviral systems used in prokaryotes. Some phages, in turn, although have small genomes can economize the use of genetic space to encode compact or ...CRISPR-Cas systems are widespread adaptive antiviral systems used in prokaryotes. Some phages, in turn, although have small genomes can economize the use of genetic space to encode compact or incomplete CRISPR-Cas systems to inhibit the host and establish infection. Phage ICP1, infecting , encodes a compact type I-F CRISPR-Cas system to suppress the antiphage mobile genetic element in the host genome. However, the mechanism by which this compact system recognizes the target DNA and executes interference remains elusive. Here, we present the electron cryo-microscopy (cryo-EM) structures of both apo- and DNA-bound ICP1 surveillance complexes (Aka Csy complex). Unlike most other type I surveillance complexes, the ICP1 Csy complex lacks the Cas11 subunit or a structurally homologous domain, which is crucial for dsDNA binding and Cas3 activation in other type I CRISPR-Cas systems. Structural and functional analyses revealed that the compact ICP1 Csy complex alone is inefficient in binding to dsDNA targets, presumably stalled at a partial R-loop conformation. The presence of Cas2/3 facilitates dsDNA binding and allows effective dsDNA target cleavage. Additionally, we found that Cas2/3 efficiently cleaved the dsDNA target presented by the ICP1 Csy complex, but not vice versa. These findings suggest a unique mechanism for target dsDNA binding and cleavage by the compact phage-derived CRISPR-Cas system.
#1: Journal: Proc.Natl.Acad.Sci.USA / Year: 2023
Title: Mechanistic insights into DNA binding and cleavage by a compact type I-F CRISPR-Cas system in bacteriophage
Authors: Zhang, M. / Peng, R. / Peng, Q. / Liu, S. / Li, Z. / Zhang, Y. / Song, H. / Yang, J. / Xing, X. / Wang, P. / Qi, J. / Gao, G.F.
History
DepositionFeb 14, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 26, 2023Provider: repository / Type: Initial release
Revision 1.1May 24, 2023Group: Database references / Category: citation / citation_author

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Csy1
B: Csy2
C: Csy3
D: Csy3
E: Csy3
F: Csy3
G: Csy3
H: Csy3
I: Csy4
M: guide-RNA


Theoretical massNumber of molelcules
Total (without water)308,18010
Polymers308,18010
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Csy1


Mass: 22841.510 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio phage ICP1_2011_A (virus) / Gene: csy1, ICP12011A_088
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: M1R2X3
#2: Protein Csy2


Mass: 29833.129 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio phage ICP1_2011_A (virus) / Gene: csy2, ICP12011A_087
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: M1QWL5
#3: Protein
Csy3


Mass: 35830.867 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio phage ICP1_2011_A (virus) / Gene: csy3, ICP12011A_086
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: M1Q7R8
#4: Protein Csy4


Mass: 21168.271 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio phage ICP1_2011_A (virus) / Gene: csy4, ICP12011A_085
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: M1R9H3
#5: RNA chain guide-RNA


Mass: 19351.408 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio phage ICP1_2011_A (virus)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: GenBank: 452118997

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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: apo-ICP1 Csy complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Vibrio phage ICP1_2011_A (virus)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 1800 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 463686 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00319285
ELECTRON MICROSCOPYf_angle_d0.57126298
ELECTRON MICROSCOPYf_dihedral_angle_d9.9833128
ELECTRON MICROSCOPYf_chiral_restr0.0422934
ELECTRON MICROSCOPYf_plane_restr0.0043316

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