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- PDB-7eln: Structure of Csy-AcrIF24-dsDNA -

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Basic information

Entry
Database: PDB / ID: 7eln
TitleStructure of Csy-AcrIF24-dsDNA
Components
  • (54-MER DNA) x 2
  • AcrIF24
  • CRISPR type I-F/YPEST-associated protein Csy2
  • CRISPR-associated protein Csy3
  • RNA (60-MER)
  • Type I-F CRISPR-associated protein Csy1
  • type I-F CRISPR-associated endoribonuclease Cas6/Csy4
KeywordsIMMUNE SYSTEM/RNA/DNA / complex / inhibitor / 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) / Uncharacterized protein / CRISPR-associated protein Csy3 / CRISPR type I-F/YPEST-associated protein Csy2
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsZhang, L. / Feng, Y.
Funding support China, 1items
OrganizationGrant numberCountry
National Science Foundation (NSF, China)31822012 China
CitationJournal: Nat Commun / Year: 2022
Title: Insights into the inhibition of type I-F CRISPR-Cas system by a multifunctional anti-CRISPR protein AcrIF24.
Authors: Lingguang Yang / Laixing Zhang / Peipei Yin / Hao Ding / Yu Xiao / Jianwei Zeng / Wenhe Wang / Huan Zhou / Qisheng Wang / Yi Zhang / Zeliang Chen / Maojun Yang / Yue Feng /
Abstract: CRISPR-Cas systems are prokaryotic adaptive immune systems and phages use anti-CRISPR proteins (Acrs) to counteract these systems. Here, we report the structures of AcrIF24 and its complex with the ...CRISPR-Cas systems are prokaryotic adaptive immune systems and phages use anti-CRISPR proteins (Acrs) to counteract these systems. Here, we report the structures of AcrIF24 and its complex with the crRNA-guided surveillance (Csy) complex. The HTH motif of AcrIF24 can bind the Acr promoter region and repress its transcription, suggesting its role as an Aca gene in self-regulation. AcrIF24 forms a homodimer and further induces dimerization of the Csy complex. Apart from blocking the hybridization of target DNA to the crRNA, AcrIF24 also induces the binding of non-sequence-specific dsDNA to the Csy complex, similar to AcrIF9, although this binding seems to play a minor role in AcrIF24 inhibitory capacity. Further structural and biochemical studies of the Csy-AcrIF24-dsDNA complexes and of AcrIF24 mutants reveal that the HTH motif of AcrIF24 and the PAM recognition loop of the Csy complex are structural elements essential for this non-specific dsDNA binding. Moreover, AcrIF24 and AcrIF9 display distinct characteristics in inducing non-specific DNA binding. Together, our findings highlight a multifunctional Acr and suggest potential wide distribution of Acr-induced non-specific DNA binding.
History
DepositionApr 12, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 20, 2022Provider: repository / Type: Initial release
Revision 1.1Apr 27, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed
Revision 1.2Jun 5, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
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: type I-F CRISPR-associated endoribonuclease Cas6/Csy4
L: CRISPR type I-F/YPEST-associated protein Csy2
M: CRISPR-associated protein Csy3
N: CRISPR-associated protein Csy3
O: CRISPR-associated protein Csy3
P: CRISPR-associated protein Csy3
Q: CRISPR-associated protein Csy3
R: CRISPR-associated protein Csy3
S: type I-F CRISPR-associated endoribonuclease Cas6/Csy4
U: AcrIF24
V: AcrIF24
J: RNA (60-MER)
T: RNA (60-MER)
A: Type I-F CRISPR-associated protein Csy1
K: 54-MER DNA
W: 54-MER DNA
X: Type I-F CRISPR-associated protein Csy1
Y: 54-MER DNA
Z: 54-MER DNA


Theoretical massNumber of molelcules
Total (without water)820,54126
Polymers820,54126
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area119250 Å2
ΔGint-390 kcal/mol
Surface area256640 Å2

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Components

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Protein , 5 types, 20 molecules BLCDEFGHMNOPQRISUVAX

#1: Protein CRISPR type I-F/YPEST-associated protein Csy2 / Type I-F CRISPR-associated protein Csy2


Mass: 36244.074 Da / Num. of mol.: 2
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
#2: Protein
CRISPR-associated protein Csy3


Mass: 37623.324 Da / Num. of mol.: 12
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
#3: Protein type I-F CRISPR-associated endoribonuclease Cas6/Csy4


Mass: 21429.477 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Production host: Escherichia coli (E. coli)
#4: Protein AcrIF24


Mass: 24995.271 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Production host: Escherichia coli (E. coli)
#6: Protein Type I-F CRISPR-associated protein Csy1


Mass: 49313.254 Da / Num. of mol.: 2
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

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RNA chain , 1 types, 2 molecules JT

#5: RNA chain RNA (60-MER)


Mass: 19265.404 Da / Num. of mol.: 2
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, 4 molecules KYWZ

#7: DNA chain 54-MER DNA


Mass: 16708.691 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Pseudomonas aeruginosa (bacteria)
#8: DNA chain 54-MER DNA


Mass: 16574.561 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Pseudomonas aeruginosa (bacteria)

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Details

Sequence detailsThe reference sequence database of chain I, S is WP_004343806.1 in GenBank. The reference sequence ...The reference sequence database of chain I, S is WP_004343806.1 in GenBank. The reference sequence database of chain U, V is WP_043084540.1 in GenBank.

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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
1Csy-AcrIF24-dsDNACOMPLEXall0MULTIPLE SOURCES
2Csy-AcrIF24COMPLEX#1-#61RECOMBINANT
3dsDNADNACOMPLEX#7-#81SYNTHETIC
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
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

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: 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
EM softwareName: RELION / Version: 3 / Category: particle selection
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1564735
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 364290 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00750249
ELECTRON MICROSCOPYf_angle_d0.81968891
ELECTRON MICROSCOPYf_dihedral_angle_d15.2968665
ELECTRON MICROSCOPYf_chiral_restr0.0527511
ELECTRON MICROSCOPYf_plane_restr0.0068620

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