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- PDB-9cp2: Post-targeting aCASCADE Type IA CRISPR_Cas Surveillance Complexes -

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Basic information

Entry
Database: PDB / ID: 9cp2
TitlePost-targeting aCASCADE Type IA CRISPR_Cas Surveillance Complexes
Components
  • CRISPR system aCascade subunit Cas5 1
  • CRISPR-associated aCascade subunit Cas7/Csa2 2
  • DNA (5'-D(P*TP*TP*TP*TP*CP*CP*TP*CP*GP*AP*A)-3')
  • RNA (27-MER)
KeywordsRNA BINDING PROTEIN/RNA/DNA / aCascade Cascade CRISPR CRISPR-Cas Cas Cas5 Cas7 crRNA TypeI-A / DNA-RNA HYBRID / RNA BINDING PROTEIN-RNA-DNA complex
Function / homology
Function and homology information


defense response to virus / RNA binding
Similarity search - Function
CRISPR-associated protein, Cas5a type / : / CRISPR-associated protein Cas7/Cst2/DevR, subtype I-a/Apern / CRISPR-associated protein Cas7/Cst2/DevR / : / CRISPR-associated negative auto-regulator DevR/Csa2 / CRISPR-associated protein Cas5, N-terminal
Similarity search - Domain/homology
: / DNA / DNA (> 10) / RNA / RNA (> 10) / CRISPR-associated aCascade subunit Cas7/Csa2 2 / CRISPR system aCascade subunit Cas5 1
Similarity search - Component
Biological speciesSaccharolobus solfataricus P2 (archaea)
Saccharolobus solfataricus (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.94 Å
AuthorsGentry, J.K. / Findlay, J.L. / Lawrence, C.M.
Funding support United States, 2items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)DBI-1828765 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P30GM140963 United States
CitationJournal: To Be Published
Title: Structures and Disassembly of Post-Targeting Type I-A CRISPR_Cas Surveillance Complexes
Authors: Gentry, J.K. / Findlay, J.L. / Lawrence, C.M.
History
DepositionJul 17, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 23, 2025Provider: repository / Type: Initial release
Revision 1.0Jul 23, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jul 23, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jul 23, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 23, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jul 23, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jul 23, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: CRISPR-associated aCascade subunit Cas7/Csa2 2
C: CRISPR-associated aCascade subunit Cas7/Csa2 2
D: CRISPR-associated aCascade subunit Cas7/Csa2 2
A: CRISPR-associated aCascade subunit Cas7/Csa2 2
G: CRISPR system aCascade subunit Cas5 1
S: RNA (27-MER)
M: DNA (5'-D(P*TP*TP*TP*TP*CP*CP*TP*CP*GP*AP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)192,61511
Polymers192,1277
Non-polymers4894
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
CRISPR-associated aCascade subunit Cas7/Csa2 2 / CRISPR-associated aCascade subunit Cas7/Csa2 / subtype I-A/Apern 2


Mass: 35308.508 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharolobus solfataricus P2 (archaea)
Gene: csa2b, cas7b, SSO1442 / Plasmid: pRSF-1b / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q97Y91
#2: Protein CRISPR system aCascade subunit Cas5 1


Mass: 27431.582 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharolobus solfataricus P2 (archaea)
Gene: cas5a, SSO1441 / Plasmid: pRSF-1b / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q97Y92
#3: RNA chain RNA (27-MER)


Mass: 20161.945 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharolobus solfataricus (archaea) / Strain: P1-16 / Plasmid: pACYCDuet-1 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: GenBank: 1935520201
#4: DNA chain DNA (5'-D(P*TP*TP*TP*TP*CP*CP*TP*CP*GP*AP*A)-3')


Mass: 3299.171 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Target DNA from the plasmid carrying the CRISPR array that copurified with the complex.
Source: (gene. exp.) Saccharolobus solfataricus (archaea) / Strain: P1-16 / Plasmid: pACYCDuet-1 / Production host: Escherichia coli BL21(DE3) (bacteria)
#5: Chemical
ChemComp-TRS / 2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / TRIS BUFFER


Mass: 122.143 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C4H12NO3 / Comment: pH buffer*YM
Has ligand of interestN
Has protein modificationN

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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: Type I-A CRISPR-Cas post-targeting surveillance subcomplex from Saccharolobus solfataricus
Type: COMPLEX / Details: Composed of Cas5, 4Cas7, crRNA and DNA / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.18 MDa / Experimental value: NO
Source (natural)Organism: Saccharolobus solfataricus (archaea) / Strain: P2
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMTris1
250 mMsodium chlorideNaCl1
SpecimenConc.: 1.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The sample was a mixture of different subcomplex assemblies of aCASCADE purified by size-exclusion chromatography
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 85 % / Chamber temperature: 277 K / Details: Blot time: 4 seconds Blot force: 5

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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: BRIGHT FIELD / Nominal magnification: 36000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Specimen holder model: FISCHIONE INSTRUMENTS DUAL AXIS TOMOGRAPHY HOLDER
Image recordingAverage exposure time: 3.7 sec. / Electron dose: 55.3 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 3901
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
7Cootmodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5400000
Details: Following blob picking and washing with 2D classification
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.94 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 379635 / Symmetry type: POINT
Atomic model buildingB value: 406 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation Coefficient
Details: Alphafold 3 homology model including RNA was rigid body fit into the density. DNA modeling was ab initio. After rigid body fitting, iterative real-space refinement and model building with ...Details: Alphafold 3 homology model including RNA was rigid body fit into the density. DNA modeling was ab initio. After rigid body fitting, iterative real-space refinement and model building with PHENIX and Coot was performed.
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE

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