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- PDB-9ebu: Wild-type EsCas13d binary complex -

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Basic information

Entry
Database: PDB / ID: 9ebu
TitleWild-type EsCas13d binary complex
Components
  • EsCas13d
  • 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: 3.06 Å
AuthorsChou, C.W. / Finkelstein, I.J.
Funding support United States, 1items
OrganizationGrant numberCountry
Welch FoundationF-1808 United States
CitationJournal: Sci Adv / Year: 2026
Title: Structural basis for target discrimination and activation by Cas13d.
Authors: Chia-Wei Chou / Selma Sinan / Hung-Che Kuo / You-Chiun Chang / Carlos Arguello / Daphne Sahaya / Rick Russell / Ilya J Finkelstein /
Abstract: CRISPR-Cas13d is increasingly used for RNA knockdowns, but off-target cleavage of near-cognate RNAs hinders its broader adoption. Here, we solve seven cryo-electron microscopy structures of wild-type ...CRISPR-Cas13d is increasingly used for RNA knockdowns, but off-target cleavage of near-cognate RNAs hinders its broader adoption. Here, we solve seven cryo-electron microscopy structures of wild-type Cas13d in complex with matched and mismatched targets. These structures reveal active, intermediate, and inactive states that illustrate a detailed activation mechanism. Upon target RNA binding, the CRISPR RNA undergoes marked conformational changes. The Helical-1 domain transitions from a docked state with the amino-terminal domain to an allosterically switched conformation that stabilizes the RNA duplex. Quantitative kinetics show that a single proximal mismatch preserves the binding rate constant but abolishes nuclease activity by trapping Cas13d in an inactive state. We also identify an active site loop in the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains that regulates substrate accessibility and can be mutated to generate both hypo- and hyperactivated variants. These findings establish the structural basis for Cas13d mismatch surveillance and provide a framework for engineering HEPN nuclease specificity and activity.
History
DepositionNov 13, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 19, 2025Provider: repository / Type: Initial release
Revision 1.0Nov 19, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Nov 19, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.1Sep 2, 2026Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: EsCas13d
B: crRNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)127,5923
Polymers127,5682
Non-polymers241
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: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / 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: #2, #1 / 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

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 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: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1

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Processing

EM softwareName: PHENIX / Version: 1.21.2_5419 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 192389 / 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: 46.15 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00488339
ELECTRON MICROSCOPYf_angle_d0.61411459
ELECTRON MICROSCOPYf_chiral_restr0.04181304
ELECTRON MICROSCOPYf_plane_restr0.00361285
ELECTRON MICROSCOPYf_dihedral_angle_d15.39781662

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