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- EMDB-27160: Type I-C Cas4-Cas1-Cas2 complex bound to a PAM/Processed prespacer -

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Basic information

Entry
Database: EMDB / ID: EMD-27160
TitleType I-C Cas4-Cas1-Cas2 complex bound to a PAM/Processed prespacer
Map data
Sample
  • Complex: Type I-C Cas4-Cas1-Cas2 complex with a PAM/Processed substrate
    • Protein or peptide: CRISPR-associated endonuclease Cas1
    • Protein or peptide: CRISPR-associated endonuclease Cas2
    • DNA: PAM/processed strand 2
    • DNA: PAM/Processed strand 1
    • Protein or peptide: CRISPR-associated exonuclease Cas4
  • Ligand: MANGANESE (II) ION
  • Ligand: IRON/SULFUR CLUSTERIron–sulfur cluster
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
CRISPR-associated exonuclease Cas4 / CRISPR-associated endonuclease Cas2 / CRISPR-associated endonuclease Cas1
Similarity search - Component
Biological speciesAlkalihalobacillus halodurans C-125 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.41 Å
AuthorsDhingra Y / Suresh SK / Juneja P / Sashital DG
Funding support United States, 2 items
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, 2022-
Header (metadata) releaseNov 2, 2022-
Map releaseNov 2, 2022-
UpdateNov 30, 2022-
Current statusNov 30, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27160.png

Downloads & links

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Map

FileDownload / File: emd_27160.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.9063 Å
Density
Contour LevelBy AUTHOR: 0.18
Minimum - Maximum-0.56222576 - 1.385946
Average (Standard dev.)7.497358e-05 (±0.02665916)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions340340340
Spacing340340340
CellA=B=C: 308.142 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_27160_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_27160_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Type I-C Cas4-Cas1-Cas2 complex with a PAM/Processed substrate

EntireName: Type I-C Cas4-Cas1-Cas2 complex with a PAM/Processed substrate
Components
  • Complex: Type I-C Cas4-Cas1-Cas2 complex with a PAM/Processed substrate
    • Protein or peptide: CRISPR-associated endonuclease Cas1
    • Protein or peptide: CRISPR-associated endonuclease Cas2
    • DNA: PAM/processed strand 2
    • DNA: PAM/Processed strand 1
    • Protein or peptide: CRISPR-associated exonuclease Cas4
  • Ligand: MANGANESE (II) ION
  • Ligand: IRON/SULFUR CLUSTERIron–sulfur cluster

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Supramolecule #1: Type I-C Cas4-Cas1-Cas2 complex with a PAM/Processed substrate

SupramoleculeName: Type I-C Cas4-Cas1-Cas2 complex with a PAM/Processed substrate
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Alkalihalobacillus halodurans C-125 (bacteria)

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Macromolecule #1: CRISPR-associated endonuclease Cas1

MacromoleculeName: CRISPR-associated endonuclease Cas1 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Alkalihalobacillus halodurans C-125 (bacteria)
Strain: ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125
Molecular weightTheoretical: 39.39334 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKKLLNTLYV TQPDTYLSLD GDNVVLLKEQ EKLGRLPLHN LEAIVGFGYT GASPALMGYC AERNISITFL TKNGRFLARV VGESRGNVV LRKTQYRISE NDQESTKIAR NFITGKVYNS KWMLERMTRE HPLRVNVEQF KATSQLLSVM MQEIRNCDSL E SLRGWEGQ ...String:
MKKLLNTLYV TQPDTYLSLD GDNVVLLKEQ EKLGRLPLHN LEAIVGFGYT GASPALMGYC AERNISITFL TKNGRFLARV VGESRGNVV LRKTQYRISE NDQESTKIAR NFITGKVYNS KWMLERMTRE HPLRVNVEQF KATSQLLSVM MQEIRNCDSL E SLRGWEGQ AAINYNKVFD QMILQQKEEF AFHGRSRRPP KDNVNAMLSF AYTLLANDVA AALETVGLDA YVGFMHQDRP GR ASLALDL MEELRGLYAD RFVLSLINRK EMTADGFYKK ENGAVLMTDE ARKTFLKAWQ TKKQEKITHP YLGEKMSWGL VPY VQALLL ARFLRGDLDE YPPFLWK

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Macromolecule #2: CRISPR-associated endonuclease Cas2

MacromoleculeName: CRISPR-associated endonuclease Cas2 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Alkalihalobacillus halodurans C-125 (bacteria)
Strain: ATCC BAA-125 / DSM 18197 / FERM 7344 / JCM 9153 / C-125
Molecular weightTheoretical: 11.038668 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
GSMLVLITYD VQTSSMGGTK RLRKVAKACQ NYGQRVQNSV FECIVDSTQL TSLKLELTSL IDEEKDSLRI YRLGNNYKTK VEHIGAKPS IDLEDPLIF

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Macromolecule #5: CRISPR-associated exonuclease Cas4

MacromoleculeName: CRISPR-associated exonuclease Cas4 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
EC number: 5' to 3' exodeoxyribonuclease (nucleoside 3'-phosphate-forming)
Source (natural)Organism: Alkalihalobacillus halodurans C-125 (bacteria)
Molecular weightTheoretical: 25.403396 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: ASNEEDRYLM LSGLQHFQFC KRQWALIHIE QQWEENVRTI EGQHLHKKAD QPFMKEKRGS KLTVRAMPIQ SKNLQISGIC DVVEFVQDS EGIELSGVSG SYKAFPVEYK RGKPKKGDED IVQLVAQAMC LEEMLVCRID KGYLFYNEIK HRVEVPITDA L RDKVVQMA ...String:
ASNEEDRYLM LSGLQHFQFC KRQWALIHIE QQWEENVRTI EGQHLHKKAD QPFMKEKRGS KLTVRAMPIQ SKNLQISGIC DVVEFVQDS EGIELSGVSG SYKAFPVEYK RGKPKKGDED IVQLVAQAMC LEEMLVCRID KGYLFYNEIK HRVEVPITDA L RDKVVQMA KEMHHYYENR HTPKVKTGPF CNNCSLQSIC LPKLMNKRSV KRYIEGRLSE

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Macromolecule #3: PAM/processed strand 2

MacromoleculeName: PAM/processed strand 2 / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 10.139498 KDa
SequenceString:
(DG)(DT)(DT)(DC)(DT)(DG)(DG)(DT)(DG)(DG) (DT)(DC)(DC)(DT)(DC)(DA)(DG)(DC)(DT)(DA) (DC)(DG)(DT)(DT)(DT)(DT)(DT)(DT)(DG) (DA)(DA)(DT)(DT)

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Macromolecule #4: PAM/Processed strand 1

MacromoleculeName: PAM/Processed strand 1 / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 9.840351 KDa
SequenceString:
(DC)(DG)(DT)(DA)(DG)(DC)(DT)(DG)(DA)(DG) (DG)(DA)(DC)(DC)(DA)(DC)(DC)(DA)(DG)(DA) (DA)(DC)(DT)(DT)(DT)(DT)(DT)(DT)(DG) (DA)(DA)(DT)

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Macromolecule #6: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: MN
Molecular weightTheoretical: 54.938 Da

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Macromolecule #7: IRON/SULFUR CLUSTER

MacromoleculeName: IRON/SULFUR CLUSTER / type: ligand / ID: 7 / Number of copies: 1 / Formula: SF4
Molecular weightTheoretical: 351.64 Da
Chemical component information

ChemComp-FS1:
IRON/SULFUR CLUSTER / Iron–sulfur cluster

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.75 mg/mL
BufferpH: 7.5
Details: 20 mM HEPES (pH 7.5), 100 mM KCl, 5% glycerol, 2 mM DTT, and 2 mM MnCl2
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2

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Image processing

Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.41 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 228000

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