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- EMDB-23840: Asymmetrical PAM-Non PAM prespacer bound Cas4/Cas1/Cas2 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-23840
TitleAsymmetrical PAM-Non PAM prespacer bound Cas4/Cas1/Cas2 complex
Map data
Sample
  • Complex: Asymmetrical PAM-PAM prespacer bound Cas4/Cas1/Cas2 complex
    • Protein or peptide: CRISPR-associated exonuclease Cas4/endonuclease Cas1 fusion
    • DNA: DNA (26-MER)
    • DNA: DNA (34-MER)
  • Protein or peptide: CRISPR-associated endoribonuclease Cas2
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: MANGANESE (II) ION
KeywordsCRISPR/Cas / Cas4 / PAM recognition / HYDROLASE-DNA complex
Function / homology
Function and homology information


5' to 3' exodeoxyribonuclease (nucleoside 3'-phosphate-forming) / CRISPR-cas system / exonuclease activity / maintenance of CRISPR repeat elements / RNA endonuclease activity / 4 iron, 4 sulfur cluster binding / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / DNA binding / metal ion binding
Similarity search - Function
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 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 endoribonuclease Cas2 / CRISPR-associated exonuclease Cas4/endonuclease Cas1 fusion
Similarity search - Component
Biological speciesGeobacter sulfurreducens (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.57 Å
AuthorsHu CY / Ke AK
CitationJournal: Nature / Year: 2021
Title: Mechanism for Cas4-assisted directional spacer acquisition in CRISPR-Cas.
Authors: Chunyi Hu / Cristóbal Almendros / Ki Hyun Nam / Ana Rita Costa / Jochem N A Vink / Anna C Haagsma / Saket R Bagde / Stan J J Brouns / Ailong Ke /
Abstract: Prokaryotes adapt to challenges from mobile genetic elements by integrating spacers derived from foreign DNA in the CRISPR array. Spacer insertion is carried out by the Cas1-Cas2 integrase complex. A ...Prokaryotes adapt to challenges from mobile genetic elements by integrating spacers derived from foreign DNA in the CRISPR array. Spacer insertion is carried out by the Cas1-Cas2 integrase complex. A substantial fraction of CRISPR-Cas systems use a Fe-S cluster containing Cas4 nuclease to ensure that spacers are acquired from DNA flanked by a protospacer adjacent motif (PAM) and inserted into the CRISPR array unidirectionally, so that the transcribed CRISPR RNA can guide target searching in a PAM-dependent manner. Here we provide a high-resolution mechanistic explanation for the Cas4-assisted PAM selection, spacer biogenesis and directional integration by type I-G CRISPR in Geobacter sulfurreducens, in which Cas4 is naturally fused with Cas1, forming Cas4/Cas1. During biogenesis, only DNA duplexes possessing a PAM-embedded 3'-overhang trigger Cas4/Cas1-Cas2 assembly. During this process, the PAM overhang is specifically recognized and sequestered, but is not cleaved by Cas4. This 'molecular constipation' prevents the PAM-side prespacer from participating in integration. Lacking such sequestration, the non-PAM overhang is trimmed by host nucleases and integrated to the leader-side CRISPR repeat. Half-integration subsequently triggers PAM cleavage and Cas4 dissociation, allowing spacer-side integration. Overall, the intricate molecular interaction between Cas4 and Cas1-Cas2 selects PAM-containing prespacers for integration and couples the timing of PAM processing with the stepwise integration to establish directionality.
History
DepositionApr 16, 2021-
Header (metadata) releaseNov 17, 2021-
Map releaseNov 17, 2021-
UpdateDec 25, 2024-
Current statusDec 25, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.264
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.264
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-7mi5
  • Surface level: 0.264
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23840.png

Downloads & links

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Map

FileDownload / File: emd_23840.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.23 Å/pix.
x 220 pix.
= 270.6 Å		1.23 Å/pix.
x 220 pix.
= 270.6 Å		1.23 Å/pix.
x 220 pix.
= 270.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.23 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.264 / Movie #1: 0.264
Minimum - Maximum-0.82422924 - 2.7157638
Average (Standard dev.)0.0008446281 (±0.048931707)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 270.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.231.231.23
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z270.600270.600270.600
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ376376376
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-0.8242.7160.001

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Supplemental data

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Sample components

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Entire : Asymmetrical PAM-PAM prespacer bound Cas4/Cas1/Cas2 complex

EntireName: Asymmetrical PAM-PAM prespacer bound Cas4/Cas1/Cas2 complex
Components
  • Complex: Asymmetrical PAM-PAM prespacer bound Cas4/Cas1/Cas2 complex
    • Protein or peptide: CRISPR-associated exonuclease Cas4/endonuclease Cas1 fusion
    • DNA: DNA (26-MER)
    • DNA: DNA (34-MER)
  • Protein or peptide: CRISPR-associated endoribonuclease Cas2
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: MANGANESE (II) ION

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Supramolecule #1: Asymmetrical PAM-PAM prespacer bound Cas4/Cas1/Cas2 complex

SupramoleculeName: Asymmetrical PAM-PAM prespacer bound Cas4/Cas1/Cas2 complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1, #3-#4 / Details: Cas4 recognizes PAM
Source (natural)Organism: Geobacter sulfurreducens (bacteria)
Molecular weightTheoretical: 300 KDa

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Macromolecule #1: CRISPR-associated exonuclease Cas4/endonuclease Cas1 fusion

MacromoleculeName: CRISPR-associated exonuclease Cas4/endonuclease Cas1 fusion
type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Geobacter sulfurreducens (bacteria) / Strain: ATCC 51573 / DSM 12127 / PCA
Molecular weightTheoretical: 62.598496 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAETDGSIPL IPVRMLNEHV YCPRLAYLMW VQGEFSHNEF TVDGVIRHRR VDAGGGVLPS ETQEDSRIHA RSVSLSSERL GITAKIDLV EGEGAYVSPV DYKRGKRPHV AGGAYEPERV QLCAQGLLLR EHGFASDGGA LYFVASRERV PVAFDDELIG R TLAAIDEM ...String:
MAETDGSIPL IPVRMLNEHV YCPRLAYLMW VQGEFSHNEF TVDGVIRHRR VDAGGGVLPS ETQEDSRIHA RSVSLSSERL GITAKIDLV EGEGAYVSPV DYKRGKRPHV AGGAYEPERV QLCAQGLLLR EHGFASDGGA LYFVASRERV PVAFDDELIG R TLAAIDEM GRTALSGTMP PPLEDSPKCP RCSLVGICLP DEVRFLSHLS VEPRPIIPAD GRGLPLYVQS PKAYVRKDGD CL VIEEERV RVAEARLGET SQVALFGNAT LTTAALHECL RREIPVTWLS YGGWFMGHTV STGHRNVETR TYQYQRSFDP ETC LNLARR WIVAKIANCR TLLRRNWRGE GDEAKAPPGL LMSLQDDMRH AMRAPSLEVL LGIEGASAGR YFQHFSRMLR GGDG EGMGF DFTTRNRRPP KDPVNALLSF AYAMLTREWT VALAAVGLDP YRGFYHQPRF GRPALALDMM EPFRPLIADS TVLMA INNG EIRTGDFVRS AGGCNLTDSA RKRFIAGFER RMEQEVTHPI FKYTISYRRL LEVQARLLTR YLSGEIPAYP NFVTR

UniProtKB: CRISPR-associated exonuclease Cas4/endonuclease Cas1 fusion

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Macromolecule #2: CRISPR-associated endoribonuclease Cas2

MacromoleculeName: CRISPR-associated endoribonuclease Cas2 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Geobacter sulfurreducens (bacteria) / Strain: ATCC 51573 / DSM 12127 / PCA
Molecular weightTheoretical: 11.190176 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MEHLYIVSYD IRNQRRWRRL FKTMHGFGCW LQLSVFQCRL DRIRIIKMEA AINEIVNHAE DHVLILDLGP AENVKPKVSS IGKTFDPIL RQAVIV

UniProtKB: CRISPR-associated endoribonuclease Cas2

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Macromolecule #3: DNA (26-MER)

MacromoleculeName: DNA (26-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Geobacter sulfurreducens (bacteria)
Molecular weightTheoretical: 11.339267 KDa
SequenceString:
(DC)(DA)(DC)(DC)(DA)(DT)(DC)(DG)(DT)(DG) (DA)(DG)(DG)(DC)(DC)(DT)(DC)(DA)(DG)(DC) (DT)(DA)(DC)(DG)(DA)(DC)(DT)(DT)(DT) (DT)(DT)(DG)(DG)(DG)(DT)(DT)(DT)

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Macromolecule #4: DNA (34-MER)

MacromoleculeName: DNA (34-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Geobacter sulfurreducens (bacteria)
Molecular weightTheoretical: 11.418327 KDa
SequenceString:
(DG)(DT)(DC)(DG)(DT)(DA)(DG)(DC)(DT)(DG) (DA)(DG)(DG)(DC)(DC)(DT)(DC)(DA)(DC)(DG) (DA)(DT)(DG)(DG)(DA)(DC)(DT)(DT)(DT) (DT)(DT)(DG)(DA)(DA)(DT)(DT)(DT)

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Macromolecule #5: IRON/SULFUR CLUSTER

MacromoleculeName: IRON/SULFUR CLUSTER / type: ligand / ID: 5 / Number of copies: 1 / Formula: SF4
Molecular weightTheoretical: 351.64 Da
Chemical component information

ChemComp-FS1:
IRON/SULFUR CLUSTER

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Macromolecule #6: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 6 / Number of copies: 6 / 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

Concentration1.0 mg/mL
BufferpH: 7.5 / Component - Concentration: 150.0 mM / Component - Formula: NaCl / Component - Name: sodium chloride / Details: with 5 mM DTT
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV / Details: 6 seconds.

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Electron microscopy

MicroscopeTFS TALOS
Specialist opticsPhase plate: VOLTA PHASE PLATE
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 1200 / Average exposure time: 0.35 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Cs: 2.7 mm / Nominal defocus min: 1.5 µm

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Image processing

Startup modelType of model: EMDB MAP
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.57 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 80000
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
Final 3D classificationSoftware - Name: cryoSPARC

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Atomic model buiding 1

RefinementProtocol: OTHER
Output model

PDB-7mi5:
Asymmetrical PAM-Non PAM prespacer bound Cas4/Cas1/Cas2 complex

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