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- PDB-8wf8: Cryo-EM structure of the PspCas13b-crRNA complex -

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Basic information

Entry
Database: PDB / ID: 8wf8
TitleCryo-EM structure of the PspCas13b-crRNA complex
Components
  • PspCas13b
  • crRNA
KeywordsRNA BINDING PROTEIN/RNA / CRISPR / RNase / RNA BINDING PROTEIN-RNA COMPLEX
Function / homologyRNA / RNA (> 10)
Function and homology information
Biological speciesPrevotella sp. (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.59 Å
AuthorsIshikawa, J. / Kato, K. / Yamashita, K. / Nishizawa, T. / Nishimasu, H.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)23KJ0720 Japan
Japan Agency for Medical Research and Development (AMED)22am0401005h0004 Japan
CitationJournal: Nat Struct Mol Biol / Year: 2025
Title: Structural insights into RNA-guided RNA editing by the Cas13b-ADAR2 complex.
Authors: Junichiro Ishikawa / Kazuki Kato / Soumya Kannan / Sae Okazaki / Soh Ishiguro / Keitaro Yamashita / Nozomu Yachie / Tomohiro Nishizawa / Feng Zhang / Hiroshi Nishimasu /
Abstract: Cas13 is an RNA-guided RNA endonuclease derived from the type VI CRISPR-Cas system, which has been used in numerous RNA-targeting technologies, such as RNA knockdown, detection and editing. The ...Cas13 is an RNA-guided RNA endonuclease derived from the type VI CRISPR-Cas system, which has been used in numerous RNA-targeting technologies, such as RNA knockdown, detection and editing. The catalytically inactive Prevotella sp. Cas13b (dPspCas13b) fused to the human adenosine deaminase acting on RNA 2 (ADAR2) deaminase domain can edit adenosine in target transcripts to inosine, in an RNA-editing technology called REPAIR (RNA editing for programmable A-to-I replacement), which has potential for gene therapy. Here we report the cryo-electron microscopy structures of the PspCas13b-guide RNA binary complex, the PspCas13b-guide RNA-target RNA ternary complex and the dPspCas13b-ADAR2-guide RNA-target RNA complex. These structures provide mechanistic insights into RNA cleavage and editing. We applied our structural insights to engineer a compact and efficient dPspCas13b-ADAR2 complex (REPAIR-mini). Overall, our findings advance the understanding of CRISPR-Cas13 effector nucleases and could enable the development of improved RNA-targeting technologies.
History
DepositionSep 19, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 19, 2025Provider: repository / Type: Initial release
Revision 1.0Mar 19, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 19, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
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Revision 1.0Mar 19, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 19, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Mar 19, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Apr 23, 2025Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / struct
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update / _em_admin.title / _struct.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: PspCas13b
B: crRNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)149,0473
Polymers149,0232
Non-polymers241
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 PspCas13b


Mass: 127930.172 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Prevotella sp. (bacteria) / Production host: Escherichia coli (E. coli)
#2: RNA chain crRNA


Mass: 21092.453 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: synthetic construct (others)
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
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 PspCas13b-crRNA complex / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Prevotella sp. (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 55.473 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.59 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 769651 / Symmetry type: POINT

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