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- EMDB-32874: DNA bound-ICP1 Csy complex -

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Basic information

Entry
Database: EMDB / ID: EMD-32874
TitleDNA bound-ICP1 Csy complex
Map data
Sample
  • Complex: target DNA bound ICP1 Csy complex
    • Protein or peptide: Csy1
    • Protein or peptide: Csy2
    • Protein or peptide: Csy3
    • RNA: guide-RNA
    • DNA: target strand DNA
    • DNA: non-target strand DNA
Function / homologyCRISPR-associated protein Csy2 / CRISPR-associated protein (Cas_Csy2) / CRISPR-associated protein Csy3 / CRISPR-associated protein (Cas_Csy3) / Csy3 / Csy2 / Csy1
Function and homology information
Biological speciesVibrio phage ICP1_2011_A (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsZhang M / Peng R
Funding support China, 1 items
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 GF
History
DepositionFeb 14, 2022-
Header (metadata) releaseApr 26, 2023-
Map releaseApr 26, 2023-
UpdateMay 24, 2023-
Current statusMay 24, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_32874.png

Downloads & links

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Map

FileDownload / File: emd_32874.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.38 Å
Density
Contour LevelBy AUTHOR: 0.035
Minimum - Maximum-0.18393533 - 0.32116425
Average (Standard dev.)0.00034564134 (±0.0071260524)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 276.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : target DNA bound ICP1 Csy complex

EntireName: target DNA bound ICP1 Csy complex
Components
  • Complex: target DNA bound ICP1 Csy complex
    • Protein or peptide: Csy1
    • Protein or peptide: Csy2
    • Protein or peptide: Csy3
    • RNA: guide-RNA
    • DNA: target strand DNA
    • DNA: non-target strand DNA

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Supramolecule #1: target DNA bound ICP1 Csy complex

SupramoleculeName: target DNA bound ICP1 Csy complex / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Vibrio phage ICP1_2011_A (virus)

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Macromolecule #1: Csy1

MacromoleculeName: Csy1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vibrio phage ICP1_2011_A (virus)
Molecular weightTheoretical: 22.84151 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MGSSHHHHHH SSGRENLYFQ GMIKEMIEDF ISKGGLIFTH SGRYTNTNNS CFIFNKNDIG VDTKVDMYTP KSAGIKNEEG ENLWQVLNK ANMFYRIYSG ELGEELQYLL KSCCTAKEDV TTLPQIYFKN GEGYDILVPI GNAHNLISGT EYLWEHKYYN T FTQKLGGS ...String:
MGSSHHHHHH SSGRENLYFQ GMIKEMIEDF ISKGGLIFTH SGRYTNTNNS CFIFNKNDIG VDTKVDMYTP KSAGIKNEEG ENLWQVLNK ANMFYRIYSG ELGEELQYLL KSCCTAKEDV TTLPQIYFKN GEGYDILVPI GNAHNLISGT EYLWEHKYYN T FTQKLGGS NPQNCTHACN KMRGGFKQFN CTPPQVEDNY NA

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Macromolecule #2: Csy2

MacromoleculeName: Csy2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vibrio phage ICP1_2011_A (virus)
Molecular weightTheoretical: 29.833129 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MGSSHHHHHH SSGRENLYFQ GMRKFIIVKN VKVDGINAKS SDITVGMPPA TTFCGLGETM SIKTGIVVKA VSYGSVKFEV RGSRFNTSV TKFAWQDRGN GGKANNNSPI QPKPLADGVF TLCFEVEWED CAEVLVDKVT NFINTARIAG GTIASFNKPF V KVAKDAEE ...String:
MGSSHHHHHH SSGRENLYFQ GMRKFIIVKN VKVDGINAKS SDITVGMPPA TTFCGLGETM SIKTGIVVKA VSYGSVKFEV RGSRFNTSV TKFAWQDRGN GGKANNNSPI QPKPLADGVF TLCFEVEWED CAEVLVDKVT NFINTARIAG GTIASFNKPF V KVAKDAEE LASVKNAMMP CYVVVDCGVE VNIFEDAVNR KLQPMVNGYK KLEKIVDNKH MRDKFTPAYL ATPTYTMIGY KM VSNVDNF DQALWQYGEN TKVKTIGGIY ND

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Macromolecule #3: Csy3

MacromoleculeName: Csy3 / type: protein_or_peptide / ID: 3 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Vibrio phage ICP1_2011_A (virus)
Molecular weightTheoretical: 35.830867 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MGSSHHHHHH SSGRENLYFQ GMTKLKAPAV LAYSRKINPT NALMFAVNWS DRDNTTAVMV GTKTVAGTQS VRGNPNDADK GNIQTVNFA NLPHNKNTLL VKYNVKFVGD VFKAELGGGE YSNTLQTALE NTDFGTLAYR YVYNIAAGRT LWRNRVGAES I ETVITVND ...String:
MGSSHHHHHH SSGRENLYFQ GMTKLKAPAV LAYSRKINPT NALMFAVNWS DRDNTTAVMV GTKTVAGTQS VRGNPNDADK GNIQTVNFA NLPHNKNTLL VKYNVKFVGD VFKAELGGGE YSNTLQTALE NTDFGTLAYR YVYNIAAGRT LWRNRVGAES I ETVITVND QTFTFSDLLV NEFDEDVDVA EIADMVAGVL SGEGFVTLKV EHYMLLGEGS EVFPSQEFVE NSKLSKQLFD LN GQAAMHD QKIGNAIRTI DTWYEDATTP IAVEPYGSVV RNGVAYRAGN KTDLFTLMDG AVNGKSLTEE DQMFVTANLI RGG VFGGGK D

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Macromolecule #4: guide-RNA

MacromoleculeName: guide-RNA / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Vibrio phage ICP1_2011_A (virus)
Molecular weightTheoretical: 19.046227 KDa
SequenceString:
CUUAAAGAGU CAACCCUUUG CUUAUCUUCC CUAUUUAAAU GUUAGCAGCC GCAUAGGCUG

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Macromolecule #5: target strand DNA

MacromoleculeName: target strand DNA / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Vibrio phage ICP1_2011_A (virus)
Molecular weightTheoretical: 18.571943 KDa
SequenceString: (DC)(DG)(DT)(DT)(DT)(DA)(DC)(DA)(DG)(DC) (DA)(DA)(DT)(DT)(DT)(DA)(DA)(DA)(DT)(DA) (DG)(DG)(DG)(DA)(DA)(DG)(DA)(DT)(DA) (DA)(DG)(DC)(DA)(DA)(DA)(DG)(DG)(DG)(DT) (DT) (DG)(DA)(DC)(DG)(DA)(DA) ...String:
(DC)(DG)(DT)(DT)(DT)(DA)(DC)(DA)(DG)(DC) (DA)(DA)(DT)(DT)(DT)(DA)(DA)(DA)(DT)(DA) (DG)(DG)(DG)(DA)(DA)(DG)(DA)(DT)(DA) (DA)(DG)(DC)(DA)(DA)(DA)(DG)(DG)(DG)(DT) (DT) (DG)(DA)(DC)(DG)(DA)(DA)(DA)(DG) (DC)(DC)(DC)(DT)(DT)(DT)(DG)(DT)(DC)(DC) (DC)(DT)

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Macromolecule #6: non-target strand DNA

MacromoleculeName: non-target strand DNA / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Vibrio phage ICP1_2011_A (virus)
Molecular weightTheoretical: 18.616916 KDa
SequenceString: (DA)(DG)(DG)(DG)(DA)(DC)(DA)(DA)(DA)(DG) (DG)(DG)(DC)(DT)(DT)(DT)(DC)(DA)(DG)(DA) (DG)(DG)(DA)(DA)(DA)(DC)(DC)(DC)(DA) (DT)(DC)(DC)(DG)(DC)(DT)(DG)(DG)(DA)(DT) (DT) (DG)(DC)(DC)(DC)(DC)(DG) ...String:
(DA)(DG)(DG)(DG)(DA)(DC)(DA)(DA)(DA)(DG) (DG)(DG)(DC)(DT)(DT)(DT)(DC)(DA)(DG)(DA) (DG)(DG)(DA)(DA)(DA)(DC)(DC)(DC)(DA) (DT)(DC)(DC)(DG)(DC)(DT)(DG)(DG)(DA)(DT) (DT) (DG)(DC)(DC)(DC)(DC)(DG)(DG)(DG) (DG)(DT)(DG)(DC)(DT)(DG)(DT)(DA)(DA)(DA) (DC)(DG)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.8 µm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6b44

Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 462158
FSC plot (resolution estimation)

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