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- PDB-9lzt: RfxCas13d-crRNA binary complex -

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Basic information

Entry
Database: PDB / ID: 9lzt
TitleRfxCas13d-crRNA binary complex
Components
  • RNA (51-MER)
  • RfxCas13d
KeywordsRNA BINDING PROTEIN/RNA / Cas13d / RNA / Complex / RNA BINDING PROTEIN-RNA complex
Function / homologyRNA / RNA (> 10)
Function and homology information
Biological speciesRuminococcus sp. XPD3002 (bacteria)
Cloning vector pgRNA-ccdB-1 (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsYang, Q.X. / Sun, Y.F.
Funding support China, 2items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)92469108 China
National Natural Science Foundation of China (NSFC)31970146 China
CitationJournal: Structure / Year: 2026
Title: Cryo-EM structure of the RfxCas13d-crRNA-off-target-RNA complex.
Authors: Qianxi Yang / Yifang Sun / Lei Sun / Tian Chi / Zhenguo Chen /
Abstract: The CRISPR-Cas system is crucial for the adaptive immune response of prokaryotes and has been widely applied for genetic engineering. Cas13d, a type VI-D CRISPR-Cas effector, functions as RNA-guided ...The CRISPR-Cas system is crucial for the adaptive immune response of prokaryotes and has been widely applied for genetic engineering. Cas13d, a type VI-D CRISPR-Cas effector, functions as RNA-guided ribonuclease and has been engineered for programmable RNA editing, which is a commonly used, active, and well-characterized small type VI editor. Here, we determined cryoelectron microscopy (cryo-EM) structures of Ruminococcus flavefaciens Cas13d in a RfxCas13d-crRNA-off-target-RNA ternary complex and RfxCas13d-crRNA binary complex at 3.10 and 3.13 Å resolution. The ternary complex consists of RfxCas13d, crRNA, and a captured short off-target ssRNA at a complex state of binding proximal mismatched RNA. RfxCas13d undergoes conformational changes with or without the off-target RNA, but the catalytic sites remain unchanged. Mg aids in stabilizing the crRNA repeat region structure, which may be crucial for RNA binding. This discovery provides the foundation for developing RfxCas13d as a mature tool and offers a framework for advancing transcriptome engineering.
History
DepositionFeb 22, 2025Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Oct 8, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: RNA (51-MER)
B: RfxCas13d
hetero molecules


Theoretical massNumber of molelcules
Total (without water)127,8593
Polymers127,8352
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: RNA chain RNA (51-MER)


Mass: 16327.809 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Cloning vector pgRNA-ccdB-1 (others)
#2: Protein RfxCas13d


Mass: 111506.922 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Ruminococcus sp. XPD3002 (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)
#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 RfxCas13d with crRNA / Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia phage EcSzw-2 (virus)
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: BRIGHT FIELD / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 58.4 e/Å2 / Film or detector model: FEI EAGLE (4k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 196037 / Symmetry type: POINT
RefinementStereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0018655
ELECTRON MICROSCOPYf_angle_d0.40111904
ELECTRON MICROSCOPYf_dihedral_angle_d23.5523406
ELECTRON MICROSCOPYf_chiral_restr0.0341344
ELECTRON MICROSCOPYf_plane_restr0.0031347

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