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- PDB-7sbb: Structure of type I-D Cascade bound to a ssRNA target -

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Basic information

Entry
Database: PDB / ID: 7sbb
TitleStructure of type I-D Cascade bound to a ssRNA target
Components
  • Cas10d
  • Cas11d
  • Cas5d
  • Cas7d
  • crRNA
  • ssRNA target
KeywordsRNA BINDING PROTEIN/RNA / CRISPR / Complex / Ribonucleoprotein complex / type I-D / type ID / type I / cyanobacteria / synechocystis / RNA binding protein / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


CRISPR-associated protein Csc1 / CRISPR associated protein Csc3 / CRISPR-associated protein Csc2 / Csc2 Crispr
Similarity search - Domain/homology
: / RNA / RNA (> 10) / Slr7013 protein / Slr7012 protein / CRISPR-associated protein Csc3
Similarity search - Component
Biological speciesSynechocystis sp. PCC 6803 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsSchwartz, E.A. / Taylor, D.W.
Funding support United States, 1items
OrganizationGrant numberCountry
Welch FoundationF-1938 United States
CitationJournal: Nat Commun / Year: 2022
Title: Structural rearrangements allow nucleic acid discrimination by type I-D Cascade.
Authors: Evan A Schwartz / Tess M McBride / Jack P K Bravo / Daniel Wrapp / Peter C Fineran / Robert D Fagerlund / David W Taylor /
Abstract: CRISPR-Cas systems are adaptive immune systems that protect prokaryotes from foreign nucleic acids, such as bacteriophages. Two of the most prevalent CRISPR-Cas systems include type I and type III. ...CRISPR-Cas systems are adaptive immune systems that protect prokaryotes from foreign nucleic acids, such as bacteriophages. Two of the most prevalent CRISPR-Cas systems include type I and type III. Interestingly, the type I-D interference proteins contain characteristic features of both type I and type III systems. Here, we present the structures of type I-D Cascade bound to both a double-stranded (ds)DNA and a single-stranded (ss)RNA target at 2.9 and 3.1 Å, respectively. We show that type I-D Cascade is capable of specifically binding ssRNA and reveal how PAM recognition of dsDNA targets initiates long-range structural rearrangements that likely primes Cas10d for Cas3' binding and subsequent non-target strand DNA cleavage. These structures allow us to model how binding of the anti-CRISPR protein AcrID1 likely blocks target dsDNA binding via competitive inhibition of the DNA substrate engagement with the Cas10d active site. This work elucidates the unique mechanisms used by type I-D Cascade for discrimination of single-stranded and double stranded targets. Thus, our data supports a model for the hybrid nature of this complex with features of type III and type I systems.
History
DepositionSep 24, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 8, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 5, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cas7d
B: Cas7d
C: Cas7d
D: Cas7d
E: Cas7d
F: Cas7d
G: Cas7d
H: Cas5d
I: Cas10d
J: Cas11d
K: Cas11d
X: ssRNA target
Z: crRNA


Theoretical massNumber of molelcules
Total (without water)455,14913
Polymers455,14913
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Gel filtration followed by SDS-PAGE analysis and mass spectrometry. Cryo-electron microscopy was performed after verification of complex assembly and purification.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 4 types, 11 molecules ABCDEFGHIJK

#1: Protein
Cas7d


Mass: 36527.109 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Gene: slr7012 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6ZEI6
#2: Protein Cas5d


Mass: 28942.748 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Gene: slr7013 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6ZEI5
#3: Protein Cas10d


Mass: 112067.508 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Gene: slr7011 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6ZEI7
#4: Protein Cas11d


Mass: 17024.494 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Gene: slr7011 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6ZEI7

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RNA chain , 2 types, 2 molecules XZ

#5: RNA chain ssRNA target


Mass: 10700.537 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#6: RNA chain crRNA


Mass: 13699.081 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis sp. PCC 6803 (bacteria) / Production host: Escherichia coli (E. coli) / References: GenBank: CP073020

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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-D Cascade bound to a ssRNA target / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.45 MDa / Experimental value: NO
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMsodium chlorideNaCl1
220 mMHEPES1
31 mMDTT1
45 %Glycerol1
SpecimenConc.: 0.16 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Monodisperse sample of elongated particles.
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: Blot force 0, blot time 4s, no wait time between sample application and blotting.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 22500 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3 sec. / Electron dose: 41.2 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2

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Processing

EM software
IDNameVersionCategory
4WarpCTF correction
7Cootmodel fitting
9RELION3initial Euler assignment
12cryoSPARC23D reconstruction
13ISOLDEmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4100000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 167000 / Symmetry type: POINT
Atomic model buildingDetails: Model was built de novo using coot then flexibly refined using ISOLDE.

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