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- EMDB-63718: CasRx-crRNA-target RNA ternary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-63718
TitleCasRx-crRNA-target RNA ternary complex
Map data
Sample
  • Complex: Ternary complex of CasRx with crRNA and target RNA
    • Protein or peptide: CasRx
    • RNA: RNA (55-MER)
    • RNA: RNA (30-MER)
  • Ligand: MAGNESIUM ION
KeywordsCas13d complex / RNA BINDING PROTEIN/RNA / RNA BINDING PROTEIN-RNA complex
Biological speciesRuminococcus sp. XPD3002 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.46 Å
AuthorsChen XY / Huang HD
Funding support1 items
OrganizationGrant numberCountry
Not funded
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
DepositionMar 12, 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_63718.png

Downloads & links

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Map

FileDownload / File: emd_63718.map.gz / Format: CCP4 / Size: 125 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.)
0.86 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: 0.05
Minimum - Maximum-0.015627204 - 1.9931246
Average (Standard dev.)0.0010452253 (±0.02209202)
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: #2

Fileemd_63718_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: #1

Fileemd_63718_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of CasRx with crRNA and target RNA

EntireName: Ternary complex of CasRx with crRNA and target RNA
Components
  • Complex: Ternary complex of CasRx with crRNA and target RNA
    • Protein or peptide: CasRx
    • RNA: RNA (55-MER)
    • RNA: RNA (30-MER)
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Ternary complex of CasRx with crRNA and target RNA

SupramoleculeName: Ternary complex of CasRx with crRNA and target RNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Ruminococcus sp. XPD3002 (bacteria)

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Macromolecule #1: CasRx

MacromoleculeName: CasRx / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Ruminococcus sp. XPD3002 (bacteria)
Molecular weightTheoretical: 111.638117 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MIEKKKSFAK GMGVKSTLVS GSKVYMTTFA EGSDARLEKI VEGDSIRSVN EGEAFSAEMA DKNAGYKIGN AKFSHPKGYA VVANNPLYT GPVQQDMLGL KETLEKRYFG ESADGNDNIC IQVIHNILDI EKILAEYITN AAYAVNNISG LDKDIIGFGK F STVYTYDE ...String:
MIEKKKSFAK GMGVKSTLVS GSKVYMTTFA EGSDARLEKI VEGDSIRSVN EGEAFSAEMA DKNAGYKIGN AKFSHPKGYA VVANNPLYT GPVQQDMLGL KETLEKRYFG ESADGNDNIC IQVIHNILDI EKILAEYITN AAYAVNNISG LDKDIIGFGK F STVYTYDE FKDPEHHRAA FNNNDKLINA IKAQYDEFDN FLDNPRLGYF GQAFFSKEGR NYIINYGNEC YDILALLSGL AH WVVANNE EESRISRTWL YNLDKNLDNE YISTLNYLYD RITNELTNSF SKNSAANVNY IAETLGINPA EFAEQYFRFS IMK EQKNLG FNITKLREVM LDRKDMSEIR KNHKVFDSIR TKVYTMMDFV IYRYYIEEDA KVAAANKSLP DNEKSLSEKD IFVI NLRGS FNDDQKDALY YDEANRIWRK LENIMHNIKE FRGNKTREYK KKDAPRLPRI LPAGRDVSAF SKLMYALTMF LDGKE INDL LTTLINKFDN IQSFLKVMPL IGVNAKFVEE YAFFKDSAKI ADELRLIKSF ARMGEPIADA RRAMYIDAIR ILGTNL SYD ELKALADTFS LDENGNKLKK GKHGMRNFII NNVISNKRFH YLIRYGDPAH LHEIAKNEAV VKFVLGRIAD IQKKQGQ NG KNQIDRYYET CIGKDKGKSV SEKVDALTKI ITGMNYDQFD KKRSVIEDTG RENAEREKFK KIISLYLTVI YHILKNIV N INARYVIGFH CVERDAQLYK EKGYDINLKK LEEKGFSSVT KLCAGIDETA PDKRKDVEKE MAERAKESID SLESANPKL YANYIKYSDE KKAEEFTRQI NREKAKTALN AYLRNTKWNV IIREDLLRID NKTCTLFANK AVALEVARYV HAYINDIAEV NSYFQLYHY IMQRIIMNER YEKSSGKVSE YFDAVNDEKK YNDRLLKLLC VPFGYCIPRF KNLSIEALFD RNEAAKFDKE K KKVSGNS

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Macromolecule #2: RNA (55-MER)

MacromoleculeName: RNA (55-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Ruminococcus sp. XPD3002 (bacteria)
Molecular weightTheoretical: 17.612586 KDa
SequenceString:
AACCCCUACC AACUGGUCGG GGUUUGAAAC GCCAACGAAC AUCAUGACAG CGCCC

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Macromolecule #3: RNA (30-MER)

MacromoleculeName: RNA (30-MER) / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Ruminococcus sp. XPD3002 (bacteria)
Molecular weightTheoretical: 9.56961 KDa
SequenceString:
UGGGCGCUGU CAUGAUGUUC GUUGGCUUCC

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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: FIELD EMISSION GUN
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: 37831
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: MAXIMUM LIKELIHOOD

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