7MIB

Half integration complex of Cas4/Cas1/Cas2 with Cas4 still on the Non-PAM side


Experimental Data Snapshot

  • Method: ELECTRON MICROSCOPY
  • Resolution: 5.80 Å
  • Aggregation State: PARTICLE 
  • Reconstruction Method: SINGLE PARTICLE 

wwPDB Validation   3D Report Full Report


This is version 1.1 of the entry. See complete history


Literature

Mechanism for Cas4-assisted directional spacer acquisition in CRISPR-Cas.

Hu, C.Almendros, C.Nam, K.H.Costa, A.R.Vink, J.N.A.Haagsma, A.C.Bagde, S.R.Brouns, S.J.J.Ke, A.

(2021) Nature 598: 515-520

  • DOI: https://doi.org/10.1038/s41586-021-03951-z
  • Primary Citation of Related Structures:  
    7MI4, 7MI5, 7MI9, 7MIB, 7MID

  • PubMed Abstract: 

    Prokaryotes adapt to challenges from mobile genetic elements by integrating spacers derived from foreign DNA in the CRISPR array 1 . Spacer insertion is carried out by the Cas1-Cas2 integrase complex 2-4 . 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) 5,6 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.


  • Organizational Affiliation

    Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA.


Macromolecules

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Entity ID: 2
MoleculeChains Sequence LengthOrganismDetailsImage
CRISPR-associated exonuclease Cas4/endonuclease Cas1 fusionB [auth A],
C [auth B],
D [auth C],
E [auth D]
559Geobacter sulfurreducens PCAMutation(s): 0 
Gene Names: cas4-cas1GSU0057
EC: 3.1 (PDB Primary Data), 3.1.12.1 (PDB Primary Data)
UniProt
Find proteins for Q74H36 (Geobacter sulfurreducens (strain ATCC 51573 / DSM 12127 / PCA))
Explore Q74H36 
Go to UniProtKB:  Q74H36
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ74H36
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  • Reference Sequence
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Entity ID: 3
MoleculeChains Sequence LengthOrganismDetailsImage
CRISPR-associated endoribonuclease Cas2F [auth E],
G [auth F]
95Geobacter sulfurreducens PCAMutation(s): 0 
Gene Names: cas2GSU0058
EC: 3.1
UniProt
Find proteins for Q74H35 (Geobacter sulfurreducens (strain ATCC 51573 / DSM 12127 / PCA))
Explore Q74H35 
Go to UniProtKB:  Q74H35
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ74H35
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  • Reference Sequence
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Entity ID: 1
MoleculeChains LengthOrganismImage
DNA (31-MER)A [auth G]31Geobacter sulfurreducens PCA
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Entity ID: 4
MoleculeChains LengthOrganismImage
DNA (64-MER)64Geobacter sulfurreducens PCA
Sequence Annotations
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  • Reference Sequence

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Entity ID: 5
MoleculeChains LengthOrganismImage
DNA (5'-D(P*CP*GP*GP*AP*AP*AP*AP*GP*AP*GP*CP*C)-3')12Geobacter sulfurreducens PCA
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Entity ID: 6
MoleculeChains LengthOrganismImage
DNA (45-MER)45Geobacter sulfurreducens PCA
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: ELECTRON MICROSCOPY
  • Resolution: 5.80 Å
  • Aggregation State: PARTICLE 
  • Reconstruction Method: SINGLE PARTICLE 
EM Software:
TaskSoftware PackageVersion
RECONSTRUCTIONRELION

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2021-11-17
    Type: Initial release
  • Version 1.1: 2024-05-29
    Changes: Data collection