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- EMDB-63531: RfxCas13d-crRNA-target RNA ternary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-63531
TitleRfxCas13d-crRNA-target RNA ternary complex
Map data
Sample
  • Complex: Ternary complex of RfxCas13d with crRNA and target RNA
    • Protein or peptide: RfxCas13d
    • RNA: RNA (51-MER)
    • RNA: RNA (5'-R(P*CP*UP*GP*UP*GP*GP*GP*AP*CP*GP*AP*GP*UP*C)-3')
  • Ligand: MAGNESIUM ION
KeywordsCas13d / RNA / Complex / RNA BINDING PROTEIN/RNA / RNA BINDING PROTEIN-RNA complex
Biological speciesEscherichia coli (E. coli) / Ruminococcus sp. XPD3002 (bacteria) / Cloning vector pgRNA-ccdB-1 (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsYang QX / Sun YF
Funding support China, 2 items
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, 2025-
Header (metadata) releaseOct 8, 2025-
Map releaseOct 8, 2025-
UpdateMar 11, 2026-
Current statusMar 11, 2026Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_63531.png

Downloads & links

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Map

FileDownload / File: emd_63531.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.064 Å
Density
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-1.0390835 - 29.694302
Average (Standard dev.)0.003810588 (±0.47913253)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 272.384 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_63531_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_63531_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of RfxCas13d with crRNA and target RNA

EntireName: Ternary complex of RfxCas13d with crRNA and target RNA
Components
  • Complex: Ternary complex of RfxCas13d with crRNA and target RNA
    • Protein or peptide: RfxCas13d
    • RNA: RNA (51-MER)
    • RNA: RNA (5'-R(P*CP*UP*GP*UP*GP*GP*GP*AP*CP*GP*AP*GP*UP*C)-3')
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Ternary complex of RfxCas13d with crRNA and target RNA

SupramoleculeName: Ternary complex of RfxCas13d with crRNA and target RNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: RfxCas13d

MacromoleculeName: RfxCas13d / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Ruminococcus sp. XPD3002 (bacteria)
Molecular weightTheoretical: 111.506922 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: IEKKKSFAKG MGVKSTLVSG SKVYMTTFAE GSDARLEKIV EGDSIRSVNE GEAFSAEMAD KNAGYKIGNA KFSHPKGYAV VANNPLYTG PVQQDMLGLK ETLEKRYFGE SADGNDNICI QVIHNILDIE KILAEYITNA AYAVNNISGL DKDIIGFGKF S TVYTYDEF ...String:
IEKKKSFAKG MGVKSTLVSG SKVYMTTFAE GSDARLEKIV EGDSIRSVNE GEAFSAEMAD KNAGYKIGNA KFSHPKGYAV VANNPLYTG PVQQDMLGLK ETLEKRYFGE SADGNDNICI QVIHNILDIE KILAEYITNA AYAVNNISGL DKDIIGFGKF S TVYTYDEF KDPEHHRAAF NNNDKLINAI KAQYDEFDNF LDNPRLGYFG QAFFSKEGRN YIINYGNECY DILALLSGLA HW VVANNEE ESRISRTWLY NLDKNLDNEY ISTLNYLYDR ITNELTNSFS KNSAANVNYI AETLGINPAE FAEQYFRFSI MKE QKNLGF NITKLREVML DRKDMSEIRK NHKVFDSIRT KVYTMMDFVI YRYYIEEDAK VAAANKSLPD NEKSLSEKDI FVIN LRGSF NDDQKDALYY DEANRIWRKL ENIMHNIKEF RGNKTREYKK KDAPRLPRIL PAGRDVSAFS KLMYALTMFL DGKEI NDLL TTLINKFDNI QSFLKVMPLI GVNAKFVEEY AFFKDSAKIA DELRLIKSFA RMGEPIADAR RAMYIDAIRI LGTNLS YDE LKALADTFSL DENGNKLKKG KHGMRNFIIN NVISNKRFHY LIRYGDPAHL HEIAKNEAVV KFVLGRIADI QKKQGQN GK NQIDRYYETC IGKDKGKSVS EKVDALTKII TGMNYDQFDK KRSVIEDTGR ENAEREKFKK IISLYLTVIY HILKNIVN I NARYVIGFHC VERDAQLYKE KGYDINLKKL EEKGFSSVTK LCAGIDETAP DKRKDVEKEM AERAKESIDS LESANPKLY ANYIKYSDEK KAEEFTRQIN REKAKTALNA YLRNTKWNVI IREDLLRIDN KTCTLFANKA VALEVARYVH AYINDIAEVN SYFQLYHYI MQRIIMNERY EKSSGKVSEY FDAVNDEKKY NDRLLKLLCV PFGYCIPRFK NLSIEALFDR NEAAKFDKEK K KVSGNS

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Macromolecule #2: RNA (51-MER)

MacromoleculeName: RNA (51-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Cloning vector pgRNA-ccdB-1 (others)
Molecular weightTheoretical: 16.327809 KDa
SequenceString:
AACCCCUACC AACUGGUCGG GGUUUGAAAC CAAAGACGAC UCGUCCCACA G

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Macromolecule #3: RNA (5'-R(P*CP*UP*GP*UP*GP*GP*GP*AP*CP*GP*AP*GP*UP*C)-3')

MacromoleculeName: RNA (5'-R(P*CP*UP*GP*UP*GP*GP*GP*AP*CP*GP*AP*GP*UP*C)-3')
type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Cloning vector pgRNA-ccdB-1 (others)
Molecular weightTheoretical: 4.518731 KDa
SequenceString:
CUGUGGGACG AGUC

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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: FEI FALCON I (4k x 4k) / Average electron dose: 58.4 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 225728
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: ANGULAR RECONSTITUTION

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