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

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Basic information

Entry
Database: PDB / ID: 7mi5
TitleAsymmetrical PAM-Non PAM prespacer bound Cas4/Cas1/Cas2 complex
Components
  • (CRISPR-associated ...) x 2
  • DNA (26-MER)
  • DNA (34-MER)
KeywordsHYDROLASE/DNA / CRISPR/Cas / Cas4 / PAM recognition / HYDROLASE-DNA complex
Function / homology
Function and homology information


5' to 3' exodeoxyribonuclease (nucleoside 3'-phosphate-forming) / exonuclease activity / maintenance of CRISPR repeat elements / RNA endonuclease activity / 4 iron, 4 sulfur cluster binding / defense response to virus / endonuclease activity / 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
: / IRON/SULFUR CLUSTER / DNA / DNA (> 10) / CRISPR-associated endoribonuclease Cas2 / CRISPR-associated exonuclease Cas4/endonuclease Cas1 fusion
Similarity search - Component
Biological speciesGeobacter sulfurreducens (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.57 Å
AuthorsHu, C.Y. / Ke, A.K.
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, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 17, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: CRISPR-associated exonuclease Cas4/endonuclease Cas1 fusion
B: CRISPR-associated exonuclease Cas4/endonuclease Cas1 fusion
C: CRISPR-associated exonuclease Cas4/endonuclease Cas1 fusion
D: CRISPR-associated exonuclease Cas4/endonuclease Cas1 fusion
E: CRISPR-associated endoribonuclease Cas2
F: CRISPR-associated endoribonuclease Cas2
G: DNA (26-MER)
H: DNA (34-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)296,21315
Polymers295,5328
Non-polymers6817
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area25310 Å2
ΔGint-168 kcal/mol
Surface area79500 Å2

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Components

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

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


Mass: 62598.496 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacter sulfurreducens (bacteria) / Strain: ATCC 51573 / DSM 12127 / PCA / Gene: cas4-cas1, GSU0057 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q74H36, Hydrolases; Acting on ester bonds, 5' to 3' exodeoxyribonuclease (nucleoside 3'-phosphate-forming)
#2: Protein CRISPR-associated endoribonuclease Cas2


Mass: 11190.176 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacter sulfurreducens (bacteria) / Strain: ATCC 51573 / DSM 12127 / PCA / Gene: cas2, GSU0058 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q74H35, Hydrolases; Acting on ester bonds

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DNA chain , 2 types, 2 molecules GH

#3: DNA chain DNA (26-MER)


Mass: 11339.267 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Geobacter sulfurreducens (bacteria)
#4: DNA chain DNA (34-MER)


Mass: 11418.327 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Geobacter sulfurreducens (bacteria)

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Non-polymers , 2 types, 7 molecules

#5: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe4S4 / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical
ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mn

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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: Asymmetrical PAM-PAM prespacer bound Cas4/Cas1/Cas2 complex
Type: COMPLEX / Details: Cas4 recognizes PAM / Entity ID: #1, #3-#4 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.3 MDa / Experimental value: YES
Source (natural)Organism: Geobacter sulfurreducens (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5 / Details: with 5 mM DTT
Buffer componentConc.: 150 mM / Name: sodium chloride / Formula: NaCl
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K / Details: 6 seconds

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Electron microscopy imaging

MicroscopyModel: TFS TALOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Image recordingAverage exposure time: 0.35 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 1200
EM imaging opticsPhase plate: VOLTA PHASE PLATE

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Processing

EM software
IDNameVersionCategory
1cryoSPARC10particle selection
2EMAN9image acquisition
4EMANCTF correction
12cryoSPARCclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.57 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 80000 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER

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