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- PDB-7mi9: Full integration complex of Cas1/Cas2 from Cas4-containing system -

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Basic information

Entry
Database: PDB / ID: 7mi9
TitleFull integration complex of Cas1/Cas2 from Cas4-containing system
Components
  • (CRISPR-associated ...) x 2
  • DNA (5'-D(P*CP*GP*GP*AP*AP*AP*AP*GP*AP*GP*CP*C)-3')
  • DNA (5'-D(P*GP*AP*AP*GP*CP*C)-3')
  • DNA (72-MER)
  • DNA (80-MER)
KeywordsHYDROLASE/DNA / CRISPR/Cas / Cas4 / PAM recognition / full integration / 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 / 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 Cas1 / PD-(D/E)XK endonuclease-like domain superfamily
Similarity search - Domain/homology
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: 3.89 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
Revision 1.1May 29, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

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Structure viewerMolecule:
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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 (80-MER)
H: DNA (72-MER)
I: DNA (5'-D(P*CP*GP*GP*AP*AP*AP*AP*GP*AP*GP*CP*C)-3')
J: DNA (5'-D(P*GP*AP*AP*GP*CP*C)-3')


Theoretical massNumber of molelcules
Total (without water)325,08610
Polymers325,08610
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area34050 Å2
ΔGint-161 kcal/mol
Surface area82020 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 (E. coli)
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 (E. coli)
References: UniProt: Q74H35, Hydrolases; Acting on ester bonds

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DNA chain , 4 types, 4 molecules GHIJ

#3: DNA chain DNA (80-MER)


Mass: 24664.730 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Geobacter sulfurreducens (bacteria)
#4: DNA chain DNA (72-MER)


Mass: 22123.074 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Geobacter sulfurreducens (bacteria)
#5: DNA chain DNA (5'-D(P*CP*GP*GP*AP*AP*AP*AP*GP*AP*GP*CP*C)-3')


Mass: 3705.445 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Geobacter sulfurreducens (bacteria)
#6: DNA chain DNA (5'-D(P*GP*AP*AP*GP*CP*C)-3')


Mass: 1818.231 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Geobacter sulfurreducens (bacteria)

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Full integration complex of Cas1/Cas2 from Cas4-containing systemCOMPLEXfull integration after Cas4 dissociationall0MULTIPLE SOURCES
2Local refinement half map (spacer side)COMPLEX1MULTIPLE SOURCES
3Local refinement half map (leader side)COMPLEX1MULTIPLE SOURCES
4low resolution map before local refinementCOMPLEX1MULTIPLE SOURCES
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.3 MDaYES
12
13
14
Source (natural)Organism: Geobacter sulfurreducens (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5 / Details: WITH 5 mM DTT
Buffer componentConc.: 150 mM / Name: sodium chloride / Formula: NaClSodium chloride
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid 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

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
2EMAN9image acquisition
4EMANCTF correction
12cryoSPARCclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.89 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 80000 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00616628
ELECTRON MICROSCOPYf_angle_d0.8423225
ELECTRON MICROSCOPYf_dihedral_angle_d42.6873467
ELECTRON MICROSCOPYf_chiral_restr0.0492526
ELECTRON MICROSCOPYf_plane_restr0.0072421

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