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- PDB-7ecw: The Csy-AcrIF14-dsDNA complex -

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Basic information

Entry
Database: PDB / ID: 7ecw
TitleThe Csy-AcrIF14-dsDNA complex
Components
  • (54-MER DNA) x 2
  • AcrIF14
  • CRISPR type I-F/YPEST-associated protein Csy2
  • CRISPR-associated protein Csy3
  • RNA (60-MER)
  • Type I-F CRISPR-associated protein Csy1
KeywordsIMMUNE SYSTEM/RNA/DNA / inhibitor / complex / IMMUNE SYSTEM / IMMUNE SYSTEM-RNA-DNA complex
Function / homology
Function and homology information


CRISPR-associated protein Csy1 / CRISPR-associated protein (Cas_Csy1) / CRISPR-associated protein Csy2 / CRISPR-associated protein (Cas_Csy2) / CRISPR-associated protein Csy3 / CRISPR-associated protein (Cas_Csy3)
Similarity search - Domain/homology
: / DNA / DNA (> 10) / RNA / RNA (> 10) / Tail protein / Uncharacterized protein / CRISPR-associated protein Csy3 / CRISPR type I-F/YPEST-associated protein Csy2
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
Moraxella phage Mcat5 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsZhang, L.X. / Feng, Y.
Funding support China, 1items
OrganizationGrant numberCountry
National Science Foundation (NSF, China)31822012 China
CitationJournal: Nucleic Acids Res / Year: 2021
Title: Insights into the dual functions of AcrIF14 during the inhibition of type I-F CRISPR-Cas surveillance complex.
Authors: Xi Liu / Laixing Zhang / Yu Xiu / Teng Gao / Ling Huang / Yongchao Xie / Lingguang Yang / Wenhe Wang / Peiyi Wang / Yi Zhang / Maojun Yang / Yue Feng /
Abstract: CRISPR-Cas systems are bacterial adaptive immune systems, and phages counteract these systems using many approaches such as producing anti-CRISPR (Acr) proteins. Here, we report the structures of ...CRISPR-Cas systems are bacterial adaptive immune systems, and phages counteract these systems using many approaches such as producing anti-CRISPR (Acr) proteins. Here, we report the structures of both AcrIF14 and its complex with the crRNA-guided surveillance (Csy) complex. Our study demonstrates that apart from interacting with the Csy complex to block the hybridization of target DNA to the crRNA, AcrIF14 also endows the Csy complex with the ability to interact with non-sequence-specific dsDNA as AcrIF9 does. Further structural studies of the Csy-AcrIF14-dsDNA complex and biochemical studies uncover that the PAM recognition loop of the Cas8f subunit of the Csy complex and electropositive patches within the N-terminal domain of AcrIF14 are essential for the non-sequence-specific dsDNA binding to the Csy-AcrIF14 complex, which is different from the mechanism of AcrIF9. Our findings highlight the prevalence of Acr-induced non-specific DNA binding and shed light on future studies into the mechanisms of such Acr proteins.
History
DepositionMar 13, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 17, 2021Provider: repository / Type: Initial release
Revision 1.1Jun 5, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

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Assembly

Deposited unit
A: Type I-F CRISPR-associated protein Csy1
B: CRISPR type I-F/YPEST-associated protein Csy2
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: AcrIF14
J: AcrIF14
M: RNA (60-MER)
N: 54-MER DNA
O: 54-MER DNA


Theoretical massNumber of molelcules
Total (without water)392,44113
Polymers392,44113
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area53800 Å2
ΔGint-170 kcal/mol
Surface area132920 Å2

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Components

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Protein , 4 types, 10 molecules ABCDEFGHIJ

#1: Protein Type I-F CRISPR-associated protein Csy1


Mass: 49313.254 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: csy1, ALP65_00954, IPC1505_30690 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A3A8DDU9
#2: Protein CRISPR type I-F/YPEST-associated protein Csy2 / Type I-F CRISPR-associated protein Csy2


Mass: 36244.074 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria)
Gene: csy2, ALP65_00953, EQH76_13810, NCTC13437_01526, PACL_0128
Production host: Escherichia coli (E. coli) / References: UniProt: B3G161
#3: Protein
CRISPR-associated protein Csy3


Mass: 37623.324 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: IPC1505_30680, IPC36_28835 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A659BSG0
#4: Protein AcrIF14


Mass: 14297.373 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Moraxella phage Mcat5 (virus) / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0R6PCL0

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RNA chain , 1 types, 1 molecules M

#5: RNA chain RNA (60-MER)


Mass: 19265.404 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Production host: Escherichia coli (E. coli) / References: GenBank: 313291946

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DNA chain , 2 types, 2 molecules NO

#6: DNA chain 54-MER DNA


Mass: 16708.691 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Production host: Escherichia coli (E. coli)
#7: DNA chain 54-MER DNA


Mass: 16574.561 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Production host: Escherichia coli (E. coli)

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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

Component
IDNameTypeEntity IDParent-IDSource
1membrane proteinCOMPLEXall0RECOMBINANT
2membrane proteinCOMPLEX#1-#3, #5-#71RECOMBINANT
3AcrIF14COMPLEX#41RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Pseudomonas aeruginosa (bacteria)287
33Moraxella phage Mcat5 (virus)1647551
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Escherichia coli (E. coli)562
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

MicroscopyModel: FEI TITAN
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: DARK FIELD
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 127454 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00723689
ELECTRON MICROSCOPYf_angle_d0.86732324
ELECTRON MICROSCOPYf_dihedral_angle_d13.8493962
ELECTRON MICROSCOPYf_chiral_restr0.0533477
ELECTRON MICROSCOPYf_plane_restr0.0064087

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