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- PDB-9m31: CasRx-crRNA binary complex -

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Basic information

Entry
Database: PDB / ID: 9m31
TitleCasRx-crRNA binary complex
Components
  • CasRx
  • RNA (51-MER)
KeywordsRNA BINDING PROTEIN/RNA / Cas13d protein complex / RNA BINDING PROTEIN-RNA complex
Function / homologyRNA / RNA (> 10)
Function and homology information
Biological speciesRuminococcus sp. XPD3002 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.86 Å
AuthorsChen, X.Y. / Huang, H.D.
Funding support China, 1items
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, 2025Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Sep 17, 2025Provider: repository / Type: Initial release
Revision 1.0Sep 17, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Sep 17, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.0Sep 17, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Sep 17, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Oct 22, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CasRx
B: RNA (51-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)128,0143
Polymers127,9902
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 CasRx


Mass: 111638.117 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Ruminococcus sp. XPD3002 (bacteria) / Production host: Escherichia coli (E. coli)
#2: RNA chain RNA (51-MER)


Mass: 16351.834 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Ruminococcus sp. XPD3002 (bacteria) / Production host: Escherichia coli (E. coli)
#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: Binary complex of CasRx with crRNA / Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Source (natural)Organism: Ruminococcus sp. XPD3002 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DARK FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.19.2_4158 / Category: model refinement
CTF correctionType: NONE
3D reconstructionResolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 194460 / Symmetry type: POINT
RefinementHighest resolution: 2.86 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0039010
ELECTRON MICROSCOPYf_angle_d0.43812374
ELECTRON MICROSCOPYf_dihedral_angle_d10.7641655
ELECTRON MICROSCOPYf_chiral_restr0.0371386
ELECTRON MICROSCOPYf_plane_restr0.0041409

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