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- EMDB-37488: Cryo-EM structure of the PspCas13b-crRNA-target RNA complex (State 2) -

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Basic information

Entry
Database: EMDB / ID: EMD-37488
TitleCryo-EM structure of the PspCas13b-crRNA-target RNA complex (State 2)
Map data
Sample
  • Complex: The PspCas13b-crRNA-target RNA complex (State 2)
    • Protein or peptide: PspCas13b
    • RNA: crRNA
    • RNA: target RNA
  • Ligand: MAGNESIUM ION
KeywordsCRISPR / RNase / RNA BINDING PROTEIN-RNA COMPLEX
Biological speciesPrevotella sp. (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.13 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_37488.png

Downloads & links

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Map

FileDownload / File: emd_37488.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 288 pix.
= 239.04 Å		0.83 Å/pix.
x 288 pix.
= 239.04 Å		0.83 Å/pix.
x 288 pix.
= 239.04 Å

Surface

Projections

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Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-1.1842163 - 1.6096293
Average (Standard dev.)-0.000038080037 (±0.03709098)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 239.04 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_37488_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_37488_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_37488_half_map_2.map
Projections & Slices
AxesZYX

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Sample components

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Entire : The PspCas13b-crRNA-target RNA complex (State 2)

EntireName: The PspCas13b-crRNA-target RNA complex (State 2)
Components
  • Complex: The PspCas13b-crRNA-target RNA complex (State 2)
    • Protein or peptide: PspCas13b
    • RNA: crRNA
    • RNA: target RNA
  • Ligand: MAGNESIUM ION

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Supramolecule #1: The PspCas13b-crRNA-target RNA complex (State 2)

SupramoleculeName: The PspCas13b-crRNA-target RNA complex (State 2) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Prevotella sp. (bacteria)

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Macromolecule #1: PspCas13b

MacromoleculeName: PspCas13b / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Prevotella sp. (bacteria)
Molecular weightTheoretical: 127.930172 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNIPALVENQ KKYFGTYSVM AMLNAQTVLD HIQKVADIEG EQNENNENLW FHPVMSHLYN AKNGYDKQPE KTMFIIERLQ SYFPFLKIM AENQREYSNG KYKQNRVEVN SNDIFEVLKR AFGVLKMYRD LTNAYKTYEE KLNDGCEFLT STEQPLSGMI N NYYTVALR ...String:
MNIPALVENQ KKYFGTYSVM AMLNAQTVLD HIQKVADIEG EQNENNENLW FHPVMSHLYN AKNGYDKQPE KTMFIIERLQ SYFPFLKIM AENQREYSNG KYKQNRVEVN SNDIFEVLKR AFGVLKMYRD LTNAYKTYEE KLNDGCEFLT STEQPLSGMI N NYYTVALR NMNERYGYKT EDLAFIQDKR FKFVKDAYGK KKSQVNTGFF LSLQDYNGDT QKKLHLSGVG IALLICLFLD KQ YINIFLS RLPIFSSYNA QSEERRIIIR SFGINSIKLP KDRIHSEKSN KSVAMDMLNE VKRCPDELFT TLSAEKQSRF RII SDDHNE VLMKRSSDRF VPLLLQYIDY GKLFDHIRFH VNMGKLRYLL KADKTCIDGQ TRVRVIEQPL NGFGRLEEAE TMRK QENGT FGNSGIRIRD FENMKRDDAN PANYPYIVDT YTHYILENNK VEMFINDKED SAPLLPVIED DRYVVKTIPS CRMST LEIP AMAFHMFLFG SKKTEKLIVD VHNRYKRLFQ AMQKEEVTAE NIASFGIAES DLPQKILDLI SGNAHGKDVD AFIRLT VDD MLTDTERRIK RFKDDRKSIR SADNKMGKRG FKQISTGKLA DFLAKDIVLF QPSVNDGENK ITGLNYRIMQ SAIAVYD SG DDYEAKQQFK LMFEKARLIG KGTTEPHPFL YKVFARSIPA NAVEFYERYL IERKFYLTGL SNEIKKGNRV DVPFIRRD Q NKWKTPAMKT LGRIYSEDLP VELPRQMFDN EIKSHLKSLP QMEGIDFNNA NVTYLIAEYM KRVLDDDFQT FYQWNRNYR YMDMLKGEYD RKGSLQHCFT SVEEREGLWK ERASRTERYR KQASNKIRSN RQMRNASSEE IETILDKRLS NSRNEYQKSE KVIRRYRVQ DALLFLLAKK TLTELADFDG ERFKLKEIMP DAEKGILSEI MPMSFTFEKG GKKYTITSEG MKLKNYGDFF V LASDKRIG NLLELVGSDI VSKEDIMEEF NKYDQCRPEI SSIVFNLEKW AFDTYPELSA RVDREEKVDF KSILKILLNN KN INKEQSD ILRKIRNAFD ANNYPDKGVV EIKALPEIAM SIKKAFGEYA IMKGSLQ

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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: 11.756089 KDa
SequenceString:
GAAUGCAGAG UAGAGGCCGC AGGAUGGUUU AGAACA

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 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

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 55.473 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: 3.13 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 60063
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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