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- EMDB-73093: cryoEM structure of CbCash filament with cA4 and tRNA Ile CAU -

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Basic information

Entry
Database: EMDB / ID: EMD-73093
TitlecryoEM structure of CbCash filament with cA4 and tRNA Ile CAU
Map dataMain Map with tRNA
Sample
  • Complex: Octnary complex of CbCash filament with tRNA leucine fragments
    • Protein or peptide: CbCash
    • RNA: tRNA Ile CAU
    • RNA: cyclic-tetra-adenylate
  • Ligand: MAGNESIUM ION
KeywordstRNA endonuclease / cA4 activation / CRISPR associated Schlafen / Csx15 / RNA BINDING PROTEIN
Biological speciesChloroflexia (bacteria) / Chloroflexi bacterium 13_1_20CM_2_59_7 (bacteria) / Escherichia coli BL21(DE3) (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.65 Å
AuthorsLiu Y / Weickert P / Strecker J
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2026
Title: Prokaryotic Schlafen proteins cleave tRNAs during type III CRISPR immunity.
Authors: Pedro Weickert / Yang Liu / Jonathan Strecker /
Abstract: Schlafen nucleases restrict viral infection in mammals by cleaving self RNAs, however, their function and mechanism in prokaryotic immunity is unknown. Here, we uncover CRISPR-associated Schlafen ...Schlafen nucleases restrict viral infection in mammals by cleaving self RNAs, however, their function and mechanism in prokaryotic immunity is unknown. Here, we uncover CRISPR-associated Schlafen (Cash) proteins containing a Schlafen domain fused to Csx15, an uncharacterized member of Rossmann-like nucleotide-binding sensors. Cash is activated by cyclic tetra-adenylate (cA₄) produced during type III CRISPR interference and induces cell toxicity by cleaving tRNAs, primarily in the T-loop. Cryo-electron microscopy structures of Chloroflexi bacterium Cash reveal an inactive dodecamer, the formation of a filament upon cA₄ binding to align catalytic interfaces, and the molecular basis of substrate recognition and cleavage in a tRNA-bound complex. We identify numerous families of prokaryotic Schlafen proteins associated with diverse antiviral defense systems and characterized by unique sensor domains. This work highlights tRNA depletion by Schlafen nucleases as an evolutionary recurring antiviral strategy and reveals mechanistic differences between Cash and human Schlafen members.
History
DepositionOct 9, 2025-
Header (metadata) releaseApr 29, 2026-
Map releaseApr 29, 2026-
UpdateJul 15, 2026-
Current statusJul 15, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_73093.png

Downloads & links

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Map

FileDownload / File: emd_73093.map.gz / Format: CCP4 / Size: 202.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain Map with tRNA
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.94 Å/pix.
x 376 pix.
= 352.688 Å		0.94 Å/pix.
x 376 pix.
= 352.688 Å		0.94 Å/pix.
x 376 pix.
= 352.688 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.938 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0351
Minimum - Maximum-0.34463012 - 0.65461814
Average (Standard dev.)0.00023898616 (±0.013795707)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions376376376
Spacing376376376
CellA=B=C: 352.68802 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_73093_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map B Map with tRNA

Fileemd_73093_half_map_1.map
AnnotationHalf Map B Map with tRNA
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map A Map with tRNA

Fileemd_73093_half_map_2.map
AnnotationHalf Map A Map with tRNA
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Octnary complex of CbCash filament with tRNA leucine fragments

EntireName: Octnary complex of CbCash filament with tRNA leucine fragments
Components
  • Complex: Octnary complex of CbCash filament with tRNA leucine fragments
    • Protein or peptide: CbCash
    • RNA: tRNA Ile CAU
    • RNA: cyclic-tetra-adenylate
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Octnary complex of CbCash filament with tRNA leucine fragments

SupramoleculeName: Octnary complex of CbCash filament with tRNA leucine fragments
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Chloroflexia (bacteria)
Molecular weightTheoretical: 280.87 kDa/nm

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Macromolecule #1: CbCash

MacromoleculeName: CbCash / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Chloroflexi bacterium 13_1_20CM_2_59_7 (bacteria)
Molecular weightTheoretical: 33.530633 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MDKLWRLLGN YVDSGRETRK VDLKREIEMK AGAGKFAKDV AAIANTIGGT GYLIIGVIDL KERKSNSPDD YVTGWNVTDL DGFSRWVNQ SLSNYCNPIP RIEIEEMTHP VCGKKIGVII IPRSLDCPHE IIRDGDGITK GMYVRRGADT FTASREELRA M MGGTDVRI ...String:
MDKLWRLLGN YVDSGRETRK VDLKREIEMK AGAGKFAKDV AAIANTIGGT GYLIIGVIDL KERKSNSPDD YVTGWNVTDL DGFSRWVNQ SLSNYCNPIP RIEIEEMTHP VCGKKIGVII IPRSLDCPHE IIRDGDGITK GMYVRRGADT FTASREELRA M MGGTDVRI VLNFLQPLTQ LNREQIGQYL NARVYEVLEP RSLPIRLDEN LSFVEQAKIL LNETGLTPEE WRTLPLIVNL PG FSPLSSI LLAEMQGRMS RLPHIIRMRP TPADRTIFEV AEVIALQDIR DKALEWSSGI

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Macromolecule #2: tRNA Ile CAU

MacromoleculeName: tRNA Ile CAU / type: rna / ID: 2 / Details: tRNA Ile CAU / Number of copies: 2
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightTheoretical: 24.445514 KDa
SequenceString:
GGCCCCUUAG CUCAGUGGUU AGAGCAGGCG ACUCAUAAUC GCUUGGUCGC UGGUUCAAGU CCAGCAGGGG CCACCA

GENBANK: GENBANK: CP053602.1

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Macromolecule #3: cyclic-tetra-adenylate

MacromoleculeName: cyclic-tetra-adenylate / type: rna / ID: 3 / Number of copies: 4
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 1.271866 KDa
SequenceString:
AAAA

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 2 / 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 statefilament

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Sample preparation

Concentration0.4 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMHEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
200.0 mMNaClsodium chloride
5.0 mMMgCl2magnicium chloride
1.0 mMTCEPtris(2-carboxyethyl)phosphine

Details: 25mM HEPES pH 7.5, 200mM NaCl, 5mM MgCl2, 1mM TCEP
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: Blot Force 15 Wait Time 0s Blot Time 3.5s.
DetailsCbCash proteins incubated with tRNA Leu at 2:1 ratio, and add cA4 at 100uM before pluging.

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Average electron dose: 47.736 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. 4.6.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.65 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.6.1) / Number images used: 305770
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: Other / Chain - Initial model type: experimental model / Details: cryoSPARC ab initio
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-9ylu:
cryoEM structure of CbCash filament with cA4 and tRNA Ile CAU

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