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- EMDB-37489: Cryo-EM structure of the dPspCas13b-ADAR2-crRNA-target RNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-37489
TitleCryo-EM structure of the dPspCas13b-ADAR2-crRNA-target RNA complex
Map data
Sample
  • Complex: The dPspCas13b-ADAR2-crRNA-target RNA complex
    • Protein or peptide: dPspCas13b-ADAR2DD
    • RNA: crRNA
    • RNA: target RNA
  • Ligand: INOSITOL HEXAKISPHOSPHATE
  • Ligand: ZINC ION
KeywordsCRISPR / RNase / RNA BINDING PROTEIN-RNA COMPLEX
Biological speciesPrevotella sp. (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.71 Å
AuthorsIshikawa J / Kato K / Yamashita K / Nishizawa T / Nishimasu H
Funding support Japan, 2 items
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, 2023-
Header (metadata) releaseMar 19, 2025-
Map releaseMar 19, 2025-
UpdateApr 23, 2025-
Current statusApr 23, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_37489.png

Downloads & links

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Map

FileDownload / File: emd_37489.map.gz / Format: CCP4 / Size: 54.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Contrast
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AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 242 pix.
= 200.86 Å		0.83 Å/pix.
x 242 pix.
= 200.86 Å		0.83 Å/pix.
x 242 pix.
= 200.86 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.075
Minimum - Maximum-0.30988157 - 0.6362783
Average (Standard dev.)0.0007969331 (±0.020641256)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin595959
Dimensions242242242
Spacing242242242
CellA=B=C: 200.86 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_37489_msk_1.map
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Half map: #2

Fileemd_37489_half_map_1.map
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Half map: #1

Fileemd_37489_half_map_2.map
Projections & Slices
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Sample components

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Entire : The dPspCas13b-ADAR2-crRNA-target RNA complex

EntireName: The dPspCas13b-ADAR2-crRNA-target RNA complex
Components
  • Complex: The dPspCas13b-ADAR2-crRNA-target RNA complex
    • Protein or peptide: dPspCas13b-ADAR2DD
    • RNA: crRNA
    • RNA: target RNA
  • Ligand: INOSITOL HEXAKISPHOSPHATE
  • Ligand: ZINC ION

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Supramolecule #1: The dPspCas13b-ADAR2-crRNA-target RNA complex

SupramoleculeName: The dPspCas13b-ADAR2-crRNA-target RNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Prevotella sp. (bacteria)

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Macromolecule #1: dPspCas13b-ADAR2DD

MacromoleculeName: dPspCas13b-ADAR2DD / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Prevotella sp. (bacteria)
Molecular weightTheoretical: 176.101406 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSYYHHHHHH DGASWSHPQF EKGSTTLEVL FQGPMNIPAL VENQKKYFGT YSVMAMLNAQ TVLDHIQKVA DIEGEQNENN ENLWFHPVM SHLYNAKNGY DKQPEKTMFI IERLQSYFPF LKIMAENQRE YSNGKYKQNR VEVNSNDIFE VLKRAFGVLK M YRDLTNAY ...String:
MSYYHHHHHH DGASWSHPQF EKGSTTLEVL FQGPMNIPAL VENQKKYFGT YSVMAMLNAQ TVLDHIQKVA DIEGEQNENN ENLWFHPVM SHLYNAKNGY DKQPEKTMFI IERLQSYFPF LKIMAENQRE YSNGKYKQNR VEVNSNDIFE VLKRAFGVLK M YRDLTNAY KTYEEKLNDG CEFLTSTEQP LSGMINNYYT VALRNMNERY GYKTEDLAFI QDKRFKFVKD AYGKKKSQVN TG FFLSLQD YNGDTQKKLH LSGVGIALLI CLFLDKQYIN IFLSRLPIFS SYNAQSEERR IIIRSFGINS IKLPKDRIHS EKS NKSVAM DMLNEVKRCP DELFTTLSAE KQSRFRIISD DHNEVLMKRS SDRFVPLLLQ YIDYGKLFDH IRFHVNMGKL RYLL KADKT CIDGQTRVRV IEQPLNGFGR LEEAETMRKQ ENGTFGNSGI RIRDFENMKR DDANPANYPY IVDTYTHYIL ENNKV EMFI NDKEDSAPLL PVIEDDRYVV KTIPSCRMST LEIPAMAFHM FLFGSKKTEK LIVDVHNRYK RLFQAMQKEE VTAENI ASF GIAESDLPQK ILDLISGNAH GKDVDAFIRL TVDDMLTDTE RRIKRFKDDR KSIRSADNKM GKRGFKQIST GKLADFL AK DIVLFQPSVN DGENKITGLN YRIMQSAIAV YDSGDDYEAK QQFKLMFEKA RLIGKGTTEP HPFLYKVFAR SIPANAVE F YERYLIERKF YLTGLSNEIK KGNRVDVPFI RRDQNKWKTP AMKTLGRIYS EDLPVELPRQ MFDNEIKSHL KSLPQMEGI DFNNANVTYL IAEYMKRVLD DDFQTFYQWN RNYRYMDMLK GEYDRKGSLQ HCFTSVEERE GLWKERASRT ERYRKQASNK IRSNRQMRN ASSEEIETIL DKRLSNSRNE YQKSEKVIRR YRVQDALLFL LAKKTLTELA DFDGERFKLK EIMPDAEKGI L SEIMPMSF TFEKGGKKYT ITSEGMKLKN YGDFFVLASD KRIGNLLELV GSDIVSKEDI MEEFNKYDQC RPEISSIVFN LE KWAFDTY PELSARVDRE EKVDFKSILK ILLNNKNINK EQSDILRKIR NAFDANNYPD KGVVEIKALP EIAMSIKKAF GEY AIMKGS LQLPPLERLT LGSQLHLPQV LADAVSRLVL GKFGDLTDNF SSPHARRKVL AGVVMTTGTD VKDAKVISVS TGTK CINGE YMSDRGLALN DCHAEIISRR SLLRFLYTQL ELYLNNKDDQ KRSIFQKSER GGFRLKENVQ FHLYISTSPC GDARI FSPH EPILEEPADR HPNRKARGQL RTKIESGQGT IPVRSNASIQ TWDGVLQGER LLTMSCSDKI ARWNVVGIQG SLLSIF VEP IYFSSIILGS LYHGDHLSRA MYQRISNIED LPPLYTLNKP LLSGISNAEA RQPGKAPNFS VNWTVGDSAI EVINATT GK DELGRASRLC KHALYCRWMR VHGKVPSHLL RSKITKPNVY HESKLAAKEY QAAKARLFTA FIKAGLGAWV EKPTEQDQ F SLT

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Macromolecule #2: crRNA

MacromoleculeName: crRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 21.092453 KDa
SequenceString:
UCUAAACCAU CCUGCGGCCU CUACUCUGCA GUUGUGGAAG GUCCAGUUUU GAGGGGCUAU UACAAC

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Macromolecule #3: target RNA

MacromoleculeName: target RNA / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 11.744078 KDa
SequenceString:
GAAUGCAGAG UAGAGGCCGC AGGAU(8AZ)GUUU AGAACA

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Macromolecule #4: INOSITOL HEXAKISPHOSPHATE

MacromoleculeName: INOSITOL HEXAKISPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: IHP
Molecular weightTheoretical: 660.035 Da
Chemical component information

ChemComp-IHP:
INOSITOL HEXAKISPHOSPHATE

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 47.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.71 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 70123
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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