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- PDB-8k0j: Crystal structure of Cas7f -

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Basic information

Entry
Database: PDB / ID: 8k0j
TitleCrystal structure of Cas7f
ComponentsCsy3
KeywordsRNA BINDING PROTEIN / DNA binding
Function / homologyCRISPR-associated protein Csy3 / CRISPR-associated protein (Cas_Csy3) / Csy3
Function and homology information
Biological speciesVibrio phage ICP1_2011_A (virus)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.365 Å
AuthorsFeng, Y. / Wang, H.
Funding support China, 1items
OrganizationGrant numberCountry
National Science Foundation (NSF, China)32171274 China
CitationJournal: Nat Chem Biol / Year: 2024
Title: Cas1 mediates the interference stage in a phage-encoded CRISPR-Cas system.
Authors: Laixing Zhang / Hao Wang / Jianwei Zeng / Xueli Cao / Zhengyu Gao / Zihe Liu / Feixue Li / Jiawei Wang / Yi Zhang / Maojun Yang / Yue Feng /
Abstract: Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are prokaryotic adaptive immune systems against invading phages and other mobile genetic elements. Notably, some phages, ...Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are prokaryotic adaptive immune systems against invading phages and other mobile genetic elements. Notably, some phages, including the Vibrio cholerae-infecting ICP1 (International Center for Diarrheal Disease Research, Bangladesh cholera phage 1), harbor CRISPR-Cas systems to counteract host defenses. Nevertheless, ICP1 Cas8f lacks the helical bundle domain essential for recruitment of helicase-nuclease Cas2/3 during target DNA cleavage and how this system accomplishes the interference stage remains unknown. Here, we found that Cas1, a highly conserved component known to exclusively work in the adaptation stage, also mediates the interference stage through connecting Cas2/3 to the DNA-bound CRISPR-associated complex for antiviral defense (Cascade; CRISPR system yersinia, Csy) of the ICP1 CRISPR-Cas system. A series of structures of Csy, Csy-dsDNA (double-stranded DNA), Cas1-Cas2/3 and Csy-dsDNA-Cas1-Cas2/3 complexes reveal the whole process of Cas1-mediated target DNA cleavage by the ICP1 CRISPR-Cas system. Together, these data support an unprecedented model in which Cas1 mediates the interference stage in a phage-encoded CRISPR-Cas system and the study also sheds light on a unique model of primed adaptation.
History
DepositionJul 9, 2023Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Jul 10, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 22, 2025Group: Database references / Structure summary / Category: citation / citation_author / pdbx_entry_details
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Csy3


Theoretical massNumber of molelcules
Total (without water)33,3991
Polymers33,3991
Non-polymers00
Water82946
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)100.516, 100.516, 54.669
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number169
Space group name H-MP61

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Components

#1: Protein Csy3 / Cas7f


Mass: 33399.301 Da / Num. of mol.: 1
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 (E. coli) / References: UniProt: M1Q7R8
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 46 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationN

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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: 2.39 Å3/Da / Density % sol: 48.47 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / Details: PEG 400, Sodium chloride

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL17U1 / Wavelength: 0.979 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Jul 13, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979 Å / Relative weight: 1
ReflectionResolution: 2.365→50 Å / Num. obs: 12986 / % possible obs: 99.7 % / Redundancy: 14.6 % / Rmerge(I) obs: 0.146 / Net I/σ(I): 32.9
Reflection shellResolution: 2.365→2.45 Å / Rmerge(I) obs: 0.155 / Num. unique obs: 1284

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Processing

Software
NameVersionClassification
PHENIX(1.13_2998: ???)refinement
HKL-3000data reduction
SCALEPACKdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.365→36.999 Å / SU ML: 0.21 / Cross valid method: FREE R-VALUE / σ(F): 1.94 / Phase error: 23.76 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2219 635 4.89 %
Rwork0.1925 --
obs0.194 12986 99.67 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.365→36.999 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2293 0 0 46 2339
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0032332
X-RAY DIFFRACTIONf_angle_d0.5033166
X-RAY DIFFRACTIONf_dihedral_angle_d5.6061373
X-RAY DIFFRACTIONf_chiral_restr0.043365
X-RAY DIFFRACTIONf_plane_restr0.003414
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.3653-2.54790.25531280.22082448X-RAY DIFFRACTION100
2.5479-2.80430.26071350.22382457X-RAY DIFFRACTION100
2.8043-3.20980.25271100.2122482X-RAY DIFFRACTION100
3.2098-4.04330.19111240.18822476X-RAY DIFFRACTION100
4.0433-36.9990.21191380.1692488X-RAY DIFFRACTION99
Refinement TLS params.Method: refined / Origin x: 7.3976 Å / Origin y: -33.7622 Å / Origin z: 5.1297 Å
111213212223313233
T0.2368 Å20.0073 Å2-0.1001 Å2-0.1871 Å2-0.0112 Å2--0.178 Å2
L3.6314 °20.6425 °20.3246 °2-3.1138 °20.9398 °2--1.5867 °2
S-0.0064 Å °0.2916 Å °-0.8701 Å °-0.1059 Å °0.0614 Å °-0.2048 Å °0.1317 Å °0.1015 Å °-0.1193 Å °
Refinement TLS groupSelection details: all

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