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- PDB-8d3m: Type I-C Cas4-Cas1-Cas2 complex bound to a PAM/Processed prespacer -

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Basic information

Entry
Database: PDB / ID: 8d3m
TitleType I-C Cas4-Cas1-Cas2 complex bound to a PAM/Processed prespacer
Components
  • (CRISPR-associated endonuclease ...) x 2
  • CRISPR-associated exonuclease Cas4
  • PAM/Processed strand 1
  • PAM/processed strand 2
KeywordsHYDROLASE/DNA / CRISPR Cas adaptation type I-C / HYDROLASE-DNA complex
Function / homology
Function and homology information


5' to 3' exodeoxyribonuclease (nucleoside 3'-phosphate-forming) / exonuclease activity / maintenance of CRISPR repeat elements / iron-sulfur cluster binding / RNA endonuclease activity / DNA endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / magnesium ion binding / protein homodimerization activity ...5' to 3' exodeoxyribonuclease (nucleoside 3'-phosphate-forming) / exonuclease activity / maintenance of CRISPR repeat elements / iron-sulfur cluster binding / RNA endonuclease activity / DNA endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / magnesium ion binding / protein homodimerization activity / DNA binding / metal ion binding
Similarity search - Function
CRISPR-associated protein Cas1, DVULG subtype / CRISPR-associated protein Cas4 / Dna2/Cas4, domain of unknown function DUF83 / Domain of unknown function DUF83 / CRISPR-associated endonuclease Cas2 / Virulence-associated protein D / CRISPR associated protein Cas2 / CRISPR associated protein Cas2 / CRISPR-associated endonuclease Cas1, N-terminal domain / CRISPR-associated protein Cas1 / CRISPR-associated endonuclease Cas1, C-terminal domain ...CRISPR-associated protein Cas1, DVULG subtype / CRISPR-associated protein Cas4 / Dna2/Cas4, domain of unknown function DUF83 / Domain of unknown function DUF83 / CRISPR-associated endonuclease Cas2 / Virulence-associated protein D / CRISPR associated protein Cas2 / CRISPR associated protein Cas2 / CRISPR-associated endonuclease Cas1, N-terminal domain / CRISPR-associated protein Cas1 / CRISPR-associated endonuclease Cas1, C-terminal domain / CRISPR associated protein Cas1 / PD-(D/E)XK endonuclease-like domain superfamily
Similarity search - Domain/homology
: / IRON/SULFUR CLUSTER / DNA / DNA (> 10) / CRISPR-associated exonuclease Cas4 / CRISPR-associated endonuclease Cas2 / CRISPR-associated endonuclease Cas1
Similarity search - Component
Biological speciesAlkalihalobacillus halodurans C-125 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.41 Å
AuthorsDhingra, Y. / Suresh, S.K. / Juneja, P. / Sashital, D.G.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM115874 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM140876 United States
CitationJournal: Mol Cell / Year: 2022
Title: PAM binding ensures orientational integration during Cas4-Cas1-Cas2-mediated CRISPR adaptation.
Authors: Yukti Dhingra / Shravanti K Suresh / Puneet Juneja / Dipali G Sashital /
Abstract: Adaptation in CRISPR-Cas systems immunizes bacteria and archaea against mobile genetic elements. In many DNA-targeting systems, the Cas4-Cas1-Cas2 complex is required for selection and processing of ...Adaptation in CRISPR-Cas systems immunizes bacteria and archaea against mobile genetic elements. In many DNA-targeting systems, the Cas4-Cas1-Cas2 complex is required for selection and processing of DNA segments containing PAM sequences prior to integration of these "prespacer" substrates as spacers in the CRISPR array. We determined cryo-EM structures of the Cas4-Cas1-Cas2 adaptation complex from the type I-C system that encodes standalone Cas1 and Cas4 proteins. The structures reveal how Cas4 specifically reads out bases within the PAM sequence and how interactions with both Cas1 and Cas2 activate Cas4 endonuclease activity. The Cas4-PAM interaction ensures tight binding between the adaptation complex and the prespacer, significantly enhancing integration of the non-PAM end into the CRISPR array and ensuring correct spacer orientation. Corroborated with our biochemical results, Cas4-Cas1-Cas2 structures with substrates representing various stages of CRISPR adaptation reveal a temporally resolved mechanism for maturation and integration of functional spacers into the CRISPR array.
History
DepositionJun 1, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 2, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 30, 2022Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR-associated endonuclease Cas1
B: CRISPR-associated endonuclease Cas1
C: CRISPR-associated endonuclease Cas1
D: CRISPR-associated endonuclease Cas1
E: CRISPR-associated endonuclease Cas2
F: CRISPR-associated endonuclease Cas2
G: PAM/processed strand 2
H: PAM/Processed strand 1
I: CRISPR-associated exonuclease Cas4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)225,44111
Polymers225,0349
Non-polymers4072
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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CRISPR-associated endonuclease ... , 2 types, 6 molecules ABCDEF

#1: Protein
CRISPR-associated endonuclease Cas1


Mass: 39393.340 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Alkalihalobacillus halodurans C-125 (bacteria)
Strain: ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125
Gene: cas1, BH0341 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q9KFX9, Hydrolases; Acting on ester bonds
#2: Protein CRISPR-associated endonuclease Cas2


Mass: 11038.668 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Alkalihalobacillus halodurans C-125 (bacteria)
Strain: ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125
Gene: cas2, BH0342 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q9KFX8, Hydrolases; Acting on ester bonds

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DNA chain , 2 types, 2 molecules GH

#3: DNA chain PAM/processed strand 2


Mass: 10139.498 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain PAM/Processed strand 1


Mass: 9840.351 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein , 1 types, 1 molecules I

#5: Protein CRISPR-associated exonuclease Cas4


Mass: 25403.396 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Alkalihalobacillus halodurans C-125 (bacteria)
Gene: cas4, E2L07_14035 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0A4Y7WTW2, 5' to 3' exodeoxyribonuclease (nucleoside 3'-phosphate-forming)

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Non-polymers , 2 types, 2 molecules

#6: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mn / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER / Iron–sulfur cluster


Mass: 351.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe4S4 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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

ComponentName: Type I-C Cas4-Cas1-Cas2 complex with a PAM/Processed substrate
Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Alkalihalobacillus halodurans C-125 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Details: 20 mM HEPES (pH 7.5), 100 mM KCl, 5% glycerol, 2 mM DTT, and 2 mM MnCl2
SpecimenConc.: 0.75 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3500 nm / Nominal defocus min: 1500 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.41 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 228000 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00515922
ELECTRON MICROSCOPYf_angle_d0.68721705
ELECTRON MICROSCOPYf_dihedral_angle_d16.6712520
ELECTRON MICROSCOPYf_chiral_restr0.0432393
ELECTRON MICROSCOPYf_plane_restr0.0072590

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