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- PDB-9ylv: Cryo-EM structure of apo CbCash dodecamer -

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Basic information

Entry
Database: PDB / ID: 9ylv
TitleCryo-EM structure of apo CbCash dodecamer
ComponentsCbCash
KeywordsRNA BINDING PROTEIN / tRNA endonuclease / cA4 activation / CRISPR associated Schlafen / Csx15
Biological speciesChloroflexia (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.86 Å
AuthorsLiu, Y. / Weickert, P. / Strecker, J.
Funding support1items
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, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 29, 2026Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: CbCash
D: CbCash
E: CbCash
F: CbCash
G: CbCash
H: CbCash
K: CbCash
I: CbCash
J: CbCash
B: CbCash
L: CbCash
A: CbCash


Theoretical massNumber of molelcules
Total (without water)402,36812
Polymers402,36812
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
CbCash


Mass: 33530.633 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chloroflexia (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria)
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: Dodecamer complex of Apo CbCash / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 401.88 kDa/nm / Experimental value: NO
Source (natural)Organism: Chloroflexia (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5 / Details: 25mM HEPES pH 7.5, 200mM NaCl, 5mM MgCl2, 1mM TCEP
Buffer component
IDConc.NameFormulaBuffer-ID
125 mM(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)HEPES1
2200 mMsodium chlorideNaCl1
35 mMmagnicium chlorideMgCl21
41 mMtris(2-carboxyethyl)phosphineTCEP1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: CbCash proteins incubated with tRNA Leu at 2:1 ratio, and add cA4 at 100uM before pluging.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: Blot Force 15 Wait Time 0s Blot Time 3.5s

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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 magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 57.04 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.6.1particle selection
2Leginonimage acquisition
4cryoSPARC4.6.1CTF correction
7cryoSPARC4.6.1model fitting
9cryoSPARC4.6.1model refinement
12cryoSPARCclassification
13cryoSPARC4.6.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 267632 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingDetails: cryoSPARC ab initio / Source name: Other / Type: experimental model

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