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- PDB-9ec8: Active state of wild-type EsCas13d ternary complex -

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Basic information

Entry
Database: PDB / ID: 9ec8
TitleActive state of wild-type EsCas13d ternary complex
Components
  • EsCas13d
  • Target RNA (matched)
  • crRNA
KeywordsRNA BINDING PROTEIN/RNA / Cas13 / CRISPR / HEPN / RNA nuclease / RNA BINDING PROTEIN-RNA complex
Function / homologyRNA / RNA (> 10) / Uncharacterized protein
Function and homology information
Biological species[Eubacterium] siraeum DSM 15702 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.07 Å
AuthorsChou, C.W. / Finkelstein, I.J.
Funding support United States, 1items
OrganizationGrant numberCountry
Welch FoundationF-1808 United States
CitationJournal: Nat Commun / Year: 2024
Title: Distinct horizontal transfer mechanisms for type I and type V CRISPR-associated transposons.
Authors: Kuang Hu / Chia-Wei Chou / Claus O Wilke / Ilya J Finkelstein /
Abstract: CASTs use both CRISPR-associated proteins and Tn7-family transposons for RNA-guided vertical and horizontal transmission. CASTs encode minimal CRISPR arrays but can't acquire new spacers. Here, we ...CASTs use both CRISPR-associated proteins and Tn7-family transposons for RNA-guided vertical and horizontal transmission. CASTs encode minimal CRISPR arrays but can't acquire new spacers. Here, we report that CASTs can co-opt defense-associated CRISPR arrays for horizontal transmission. A bioinformatic analysis shows that CASTs co-occur with defense-associated CRISPR systems, with the highest prevalence for type I-B and type V CAST sub-types. Using an E. coli quantitative transposition assay and in vitro reconstitution, we show that CASTs can use CRISPR RNAs from these defense systems. A high-resolution structure of the type I-F CAST-Cascade in complex with a type III-B CRISPR RNA reveals that Cas6 recognizes direct repeats via sequence-independent π - π interactions. In addition to using heterologous CRISPR arrays, type V CASTs can also transpose via an unguided mechanism, even when the S15 co-factor is over-expressed. Over-expressing S15 and the trans-activating CRISPR RNA or a single guide RNA reduces, but does not abrogate, off-target integration for type V CASTs. Our findings suggest that some CASTs may exploit defense-associated CRISPR arrays and that this fact must be considered when porting CASTs to heterologous bacterial hosts. More broadly, this work will guide further efforts to engineer the activity and specificity of CASTs for gene editing applications.
History
DepositionNov 13, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 6, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: EsCas13d
B: crRNA
C: Target RNA (matched)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)137,2055
Polymers137,1573
Non-polymers492
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein EsCas13d


Mass: 110828.938 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) [Eubacterium] siraeum DSM 15702 (bacteria)
Gene: EUBSIR_02687 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: B0MS50
#2: RNA chain crRNA


Mass: 16739.029 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) [Eubacterium] siraeum DSM 15702 (bacteria)
Production host: Escherichia coli BL21(DE3) (bacteria)
#3: RNA chain Target RNA (matched)


Mass: 9588.762 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) [Eubacterium] siraeum DSM 15702 (bacteria)
Production host: Escherichia coli BL21(DE3) (bacteria)
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
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: The active state of EsCas13d protein with crRNA and matched target
Type: COMPLEX / Entity ID: #1-#3 / Source: NATURAL
Molecular weightValue: 0.135 MDa / Experimental value: NO
Source (natural)Organism: [Eubacterium] siraeum DSM 15702 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenConc.: 0.135 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Image recordingElectron dose: 80 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7786

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Processing

EM software
IDNameCategory
7Cootmodel fitting
9PHENIXmodel refinement
10ISOLDEmodel refinement
11cryoSPARCinitial Euler assignment
12cryoSPARCfinal Euler assignment
13cryoSPARCclassification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.07 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 176095 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 93.24 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00338967
ELECTRON MICROSCOPYf_angle_d0.552312429
ELECTRON MICROSCOPYf_chiral_restr0.0371435
ELECTRON MICROSCOPYf_plane_restr0.00331312
ELECTRON MICROSCOPYf_dihedral_angle_d16.70222035

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