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- EMDB-27161: Type I-C Cas4-Cas1-Cas2 complex bound to half-site integration in... -

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Basic information

Entry
Database: EMDB / ID: EMD-27161
TitleType I-C Cas4-Cas1-Cas2 complex bound to half-site integration intermediate (HSI)
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: HSI strand 2- integrated prespacer strand plus repeat
    • DNA: HSI strand 2
    • Protein or peptide: CRISPR-associated exonuclease Cas4
    • DNA: HSI strand 3 bottom strand leader-repeat
    • DNA: HSI strand 4 top leader strand
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: MANGANESE (II) ION
KeywordsCRISPR 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 / hydrolase activity / magnesium ion binding ...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 / hydrolase activity / 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, 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: 4.26 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-
UpdateDec 25, 2024-
Current statusDec 25, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27161.png

Downloads & links

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Map

FileDownload / File: emd_27161.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.91 Å/pix.
x 340 pix.
= 308.142 Å		0.91 Å/pix.
x 340 pix.
= 308.142 Å		0.91 Å/pix.
x 340 pix.
= 308.142 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.9063 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.11
Minimum - Maximum-0.3748095 - 1.1182511
Average (Standard dev.)0.00037993272 (±0.02031764)
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_27161_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_27161_half_map_2.map
Projections & Slices
AxesZYX

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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: HSI strand 2- integrated prespacer strand plus repeat
    • DNA: HSI strand 2
    • Protein or peptide: CRISPR-associated exonuclease Cas4
    • DNA: HSI strand 3 bottom strand leader-repeat
    • DNA: HSI strand 4 top leader strand
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: MANGANESE (II) ION

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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 / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
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: 39.665602 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GAGSMKKLLN TLYVTQPDTY LSLDGDNVVL LKEQEKLGRL PLHNLEAIVG FGYTGASPAL MGYCAERNIS ITFLTKNGRF LARVVGESR GNVVLRKTQY RISENDQEST KIARNFITGK VYNSKWMLER MTREHPLRVN VEQFKATSQL LSVMMQEIRN C DSLESLRG ...String:
GAGSMKKLLN TLYVTQPDTY LSLDGDNVVL LKEQEKLGRL PLHNLEAIVG FGYTGASPAL MGYCAERNIS ITFLTKNGRF LARVVGESR GNVVLRKTQY RISENDQEST KIARNFITGK VYNSKWMLER MTREHPLRVN VEQFKATSQL LSVMMQEIRN C DSLESLRG WEGQAAINYN KVFDQMILQQ KEEFAFHGRS RRPPKDNVNA MLSFAYTLLA NDVAAALETV GLDAYVGFMH QD RPGRASL ALDLMEELRG LYADRFVLSL INRKEMTADG FYKKENGAVL MTDEARKTFL KAWQTKKQEK ITHPYLGEKM SWG LVPYVQ ALLLARFLRG DLDEYPPFLW K

UniProtKB: CRISPR-associated endonuclease Cas1

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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: 11.166798 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
GAGSMLVLIT YDVQTSSMGG TKRLRKVAKA CQNYGQRVQN SVFECIVDST QLTSLKLELT SLIDEEKDSL RIYRLGNNYK TKVEHIGAK PSIDLEDPLI F

UniProtKB: CRISPR-associated endonuclease Cas2

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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

UniProtKB: CRISPR-associated exonuclease Cas4

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Macromolecule #3: HSI strand 2- integrated prespacer strand plus repeat

MacromoleculeName: HSI strand 2- integrated prespacer strand plus repeat / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 17.915424 KDa
SequenceString: (DG)(DT)(DA)(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) (DT)(DC)(DG)(DC)(DA)(DC)(DT)(DC)(DT)(DT) (DC) (DA)(DT)(DG)(DG)(DG)(DT) ...String:
(DG)(DT)(DA)(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) (DT)(DC)(DG)(DC)(DA)(DC)(DT)(DC)(DT)(DT) (DC) (DA)(DT)(DG)(DG)(DG)(DT)(DG)(DC) (DG)(DT)(DG)(DG)(DA)(DT)(DT)(DG)(DA)(DA)

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Macromolecule #4: HSI strand 2

MacromoleculeName: HSI strand 2 / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 11.048107 KDa
SequenceString:
(DC)(DG)(DT)(DA)(DG)(DC)(DT)(DG)(DA)(DG) (DG)(DA)(DC)(DC)(DA)(DC)(DC)(DA)(DG)(DT) (DA)(DC)(DT)(DT)(DT)(DT)(DT)(DT)(DG) (DA)(DA)(DT)(DT)(DT)(DT)(DT)

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Macromolecule #6: HSI strand 3 bottom strand leader-repeat

MacromoleculeName: HSI strand 3 bottom strand leader-repeat / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 12.583116 KDa
SequenceString:
(DT)(DT)(DC)(DA)(DA)(DT)(DC)(DC)(DA)(DC) (DG)(DC)(DA)(DC)(DC)(DC)(DA)(DT)(DG)(DA) (DA)(DG)(DA)(DG)(DG)(DC)(DG)(DG)(DA) (DC)(DA)(DG)(DC)(DG)(DA)(DA)(DA)(DA)(DT) (DC) (DC)

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Macromolecule #7: HSI strand 4 top leader strand

MacromoleculeName: HSI strand 4 top leader strand / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.355194 KDa
SequenceString:
(DG)(DG)(DA)(DT)(DT)(DT)(DT)(DC)(DG)(DC) (DT)

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Macromolecule #8: IRON/SULFUR CLUSTER

MacromoleculeName: IRON/SULFUR CLUSTER / type: ligand / ID: 8 / Number of copies: 1 / Formula: SF4
Molecular weightTheoretical: 351.64 Da
Chemical component information

ChemComp-FS1:
IRON/SULFUR CLUSTER

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Macromolecule #9: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 9 / Number of copies: 1 / Formula: MN
Molecular weightTheoretical: 54.938 Da

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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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

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

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.26 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 182000
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE

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