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- EMDB-73094: Cryo-EM structure of apo CbCash dodecamer -

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Basic information

Entry
Database: EMDB / ID: EMD-73094
TitleCryo-EM structure of apo CbCash dodecamer
Map dataApo_cash map
Sample
  • Complex: Dodecamer complex of Apo CbCash
    • Protein or peptide: CbCash
KeywordstRNA endonuclease / cA4 activation / CRISPR associated Schlafen / Csx15 / RNA BINDING PROTEIN
Biological speciesChloroflexia (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.86 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_73094.png

Downloads & links

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Map

FileDownload / File: emd_73094.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationApo_cash map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.65 Å/pix.
x 440 pix.
= 285.56 Å		0.65 Å/pix.
x 440 pix.
= 285.56 Å		0.65 Å/pix.
x 440 pix.
= 285.56 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.649 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.008
Minimum - Maximum-0.09300131 - 0.17797479
Average (Standard dev.)0.0006174241 (±0.003924888)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 285.56 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_73094_msk_1.map
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_73094_half_map_1.map
Projections & Slices
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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_73094_half_map_2.map
Projections & Slices
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Sample components

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Entire : Dodecamer complex of Apo CbCash

EntireName: Dodecamer complex of Apo CbCash
Components
  • Complex: Dodecamer complex of Apo CbCash
    • Protein or peptide: CbCash

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Supramolecule #1: Dodecamer complex of Apo CbCash

SupramoleculeName: Dodecamer complex of Apo CbCash / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Chloroflexia (bacteria)
Molecular weightTheoretical: 401.88 kDa/nm

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Macromolecule #1: CbCash

MacromoleculeName: CbCash / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Chloroflexia (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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2 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
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: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 57.04 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: 130000
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: D2 (2x2 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.6.1) / Number images used: 267632
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-9ylv:
Cryo-EM structure of apo CbCash dodecamer

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