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- EMDB-63598: DjCas13d-crRNA-target RNA2 ternary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-63598
TitleDjCas13d-crRNA-target RNA2 ternary complex
Map data
Sample
  • Complex: Ternary complex of DjCas13d with crRNA and target RNA2
    • Protein or peptide: DjCas13d
    • RNA: RNA (51-MER)
    • RNA: RNA (34-MER)
  • Ligand: MAGNESIUM ION
KeywordsCas13d prorein complex / RNA BINDING PROTEIN/RNA / RNA BINDING PROTEIN-RNA complex
Biological speciesRuminococcus sp. UBA7013 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.46 Å
AuthorsChen XY / Huang HD
Funding support China, 1 items
OrganizationGrant numberCountry
Not funded China
CitationJournal: Nucleic Acids Res / Year: 2025
Title: Mechanistic insights into Cas13d enzymes from cryo-EM structures of CasRx and DjCas13d.
Authors: Xiaoyan Chen / Yongru He / Maochao Guo / Shiyu Liu / Yue Li / Fuxing Zeng / Chongyuan Wang / Kai Yuan / Hongda Huang /
Abstract: CasRx and its engineered variants have emerged as powerful RNA-targeting tools, exhibiting high specificity, robust efficiency, and minimal trans-cleavage activity. Recently, DjCas13d was identified ...CasRx and its engineered variants have emerged as powerful RNA-targeting tools, exhibiting high specificity, robust efficiency, and minimal trans-cleavage activity. Recently, DjCas13d was identified as a promising alternative, offering even lower trans-cleavage activity while retaining comparable cis-cleavage efficiency. Despite their broad utility in biotechnology and therapeutic development, the molecular mechanisms governing substrate recognition and activation in these functionally relevant Cas13d enzymes remain incompletely understood. Here, we present comparative structural and biochemical analyses of CasRx and DjCas13d. Using cryogenic electron microscopy, we determined structures of both enzymes in binary (protein-crRNA) and ternary (protein-crRNA-target RNA) states, and additionally solved the apo structure of DjCas13d. Biochemical assays revealed that both enzymes exhibit similar cis-cleavage activity, whereas DjCas13d shows substantially reduced trans-cleavage activity relative to CasRx. Structural comparisons uncovered key conformational changes linked to target RNA engagement and catalytic activation, providing mechanistic insight into their distinct cleavage behaviors. Furthermore, structure-guided mutagenesis yielded several CasRx variants that achieve a favorable balance between reduced trans-cleavage activity and preserved cis-cleavage efficiency, representing valuable starting points for further optimization. Together, these findings advance our mechanistic understanding of Cas13 enzymes and provide a structural framework for the rational design of RNA-targeting technologies.
History
DepositionFeb 28, 2025-
Header (metadata) releaseSep 17, 2025-
Map releaseSep 17, 2025-
UpdateOct 22, 2025-
Current statusOct 22, 2025Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_63598.png

Downloads & links

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Map

FileDownload / File: emd_63598.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 320 pix.
= 273.6 Å		0.86 Å/pix.
x 320 pix.
= 273.6 Å		0.86 Å/pix.
x 320 pix.
= 273.6 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.855 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.01
Minimum - Maximum-0.0017547925 - 2.0328581
Average (Standard dev.)0.00078592764 (±0.020658601)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 273.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_63598_half_map_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_63598_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of DjCas13d with crRNA and target RNA2

EntireName: Ternary complex of DjCas13d with crRNA and target RNA2
Components
  • Complex: Ternary complex of DjCas13d with crRNA and target RNA2
    • Protein or peptide: DjCas13d
    • RNA: RNA (51-MER)
    • RNA: RNA (34-MER)
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Ternary complex of DjCas13d with crRNA and target RNA2

SupramoleculeName: Ternary complex of DjCas13d with crRNA and target RNA2
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Ruminococcus sp. UBA7013 (bacteria)

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Macromolecule #1: DjCas13d

MacromoleculeName: DjCas13d / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Ruminococcus sp. UBA7013 (bacteria)
Molecular weightTheoretical: 102.144891 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKKQKSKKTV SKTSGLKEAL SVQGTVIMTS FGKGNMANLS YKIPSSQKPQ NLNSSAGLKN VEVSGKKIKF QGRHPKIATT DNPLFKPQP GMDLLCLKDK LEMHYFGKTF DDNIHIQLIY QILDIEKILA VHVNNIVFTL DNVLHPQKEE LTEDFIGAGG W RINLDYQT ...String:
MKKQKSKKTV SKTSGLKEAL SVQGTVIMTS FGKGNMANLS YKIPSSQKPQ NLNSSAGLKN VEVSGKKIKF QGRHPKIATT DNPLFKPQP GMDLLCLKDK LEMHYFGKTF DDNIHIQLIY QILDIEKILA VHVNNIVFTL DNVLHPQKEE LTEDFIGAGG W RINLDYQT LRGQTNKYDR FKNYIKRKEL LYFGEAFYHE NERRYEEDIF AILTLLSALA QFCFASDLSS DESDHVNSFW LY QLEDQLS DEFKETLSIL WEEVTERIDS EFLKTNTVNL HILCHVFPKE SKETIVRAYY EFLIKKSFKN MGFSIKKLRE IML EQSDLK SFKEDKYNSV RAKLYKLFDF IITYYYDHHA FEKEALVSSL RSSLTEENKE EIYIKTARTL ASALGADFKK AAAD VNAKN IRDYQKKAND YRISFEDIKI GNTGIGYFSE LIYMLTLLLD GKEINDLLTT LINKFDNIIS FIDILKKLNL EFKFK PEYA DFFNMTNCRY TLEELRVINS IARMQKPSAD ARKIMYRDAL RILGMDNRPD EEIDRELERT MPVGADGKFI KGKQGF RNF IASNVIESSR FHYLVRYNNP HKTRTLVKNP NVVKFVLEGI PETQIKRYFD VCKGQEIPPT SDKSAQIDVL ARIISSV DY KIFEDVPQSA KINKDDPSRN FSDALKKQRY QAIVSLYLTV MYLITKNLVY VNSRYVIAFH CLERDAFLHG VTLPKMNK K IVYSQLTTHL LTDKNYTTYG HLKNQKGHRK WYVLVKNNLQ NSDITAVSSF ANIVAAISVV RNSNEYISGI GELHSYFEL YHYLVQSMIA KNNWYDTSHQ PKTAEYLNNL KKHHTYCKDF VKAYCIPFGY VVPRYKNLTI NELFDRNNPN PEPKEEV

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Macromolecule #2: RNA (51-MER)

MacromoleculeName: RNA (51-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Ruminococcus sp. UBA7013 (bacteria)
Molecular weightTheoretical: 16.382896 KDa
SequenceString:
CAACCCCGUA AAAAUACGGG GUUCUGAAAC GCCAACGAAC AUCAUGACAG C

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Macromolecule #3: RNA (34-MER)

MacromoleculeName: RNA (34-MER) / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Ruminococcus sp. UBA7013 (bacteria)
Molecular weightTheoretical: 10.872352 KDa
SequenceString:
UGUGGGCGCU GUCAUGAUGU UCGUUGGCUU CCUG

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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: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: OTHER
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DARK FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: NONE
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.46 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 76678
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: MAXIMUM LIKELIHOOD

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