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- PDB-7wkp: ICP1 Csy4 -

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Basic information

Entry
Database: PDB / ID: 7wkp
TitleICP1 Csy4
Components
  • 3' stem loop crRNA
  • Csy4
KeywordsRNA BINDING PROTEIN/RNA / Complex / RNA binding protein / RNA BINDING PROTEIN-RNA complex
Function / homologyCRISPR-associated endoribonuclease Cas6/Csy4, subtype I-F/YPEST / CRISPR-associated endoribonuclease Cas6/Csy4, subtype I-F/YPEST superfamily / CRISPR-associated protein (Cas_Csy4) / maintenance of CRISPR repeat elements / endonuclease activity / : / RNA / RNA (> 10) / Csy4
Function and homology information
Biological speciesVibrio phage ICP1_2011_A (virus)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2 Å
AuthorsZhang, M. / Peng, R.
Funding support China, 1items
OrganizationGrant numberCountry
Chinese Academy of Sciences 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
DepositionJan 10, 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
Revision 1.2Nov 29, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Csy4
B: 3' stem loop crRNA


Theoretical massNumber of molelcules
Total (without water)40,2142
Polymers40,2142
Non-polymers00
Water3,909217
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3730 Å2
ΔGint-28 kcal/mol
Surface area11140 Å2
MethodPISA
Unit cell
Length a, b, c (Å)57.892, 57.892, 355.786
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number178
Space group name H-MP6122
Space group name HallP612(x,y,z+5/12)

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Components

#1: 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
#2: RNA chain 3' stem loop crRNA


Mass: 19046.227 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
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 217 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.14 Å3/Da / Density % sol: 42.52 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop
Details: 0.4M Sodium thiocyanate, 0.1M Sodium acetate pH 4.0, 16% w/v PEG 4000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL19U1 / Wavelength: 1.03923 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Apr 1, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.03923 Å / Relative weight: 1
ReflectionResolution: 1.996→31.04 Å / Num. obs: 25046 / % possible obs: 98.4 % / Redundancy: 25.9 % / Biso Wilson estimate: 25.24 Å2 / Rmerge(I) obs: 0.145 / Net I/σ(I): 26.314
Reflection shellResolution: 1.996→2.067 Å / Rmerge(I) obs: 1.88 / Num. unique obs: 2101

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
SCALAdata scaling
PDB_EXTRACT3.27data extraction
HKL-2000data reduction
CNSphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2XLI

2xli


Resolution: 2→31.04 Å / SU ML: 0.2028 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 23.2713
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2438 1257 5.02 %
Rwork0.214 23786 -
obs0.2156 25043 98.44 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 29.6 Å2
Refinement stepCycle: LAST / Resolution: 2→31.04 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1304 433 0 217 1954
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00221811
X-RAY DIFFRACTIONf_angle_d0.52442537
X-RAY DIFFRACTIONf_chiral_restr0.0411311
X-RAY DIFFRACTIONf_plane_restr0.0032242
X-RAY DIFFRACTIONf_dihedral_angle_d10.2546420
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2-2.080.30581130.28672240X-RAY DIFFRACTION86.35
2.08-2.170.26821290.26452566X-RAY DIFFRACTION99.26
2.17-2.280.3241290.26032639X-RAY DIFFRACTION99.96
2.28-2.430.30391540.25342627X-RAY DIFFRACTION100
2.43-2.620.26371320.23682629X-RAY DIFFRACTION100
2.62-2.880.2671480.22152664X-RAY DIFFRACTION100
2.88-3.290.27241350.19552684X-RAY DIFFRACTION100
3.29-4.150.18771630.17752762X-RAY DIFFRACTION100
4.15-31.040.21361540.1992975X-RAY DIFFRACTION99.9

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