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- PDB-20yv: Cryo-EM structure of SspE from E.coli -

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Basic information

Entry
Database: PDB / ID: 20yv
TitleCryo-EM structure of SspE from E.coli
ComponentsDUF262 domain-containing protein
KeywordsDNA BINDING PROTEIN / Nuclease / DNA phosphorothioate / defense system
Function / homologyDomain of unknown function DUF1524 / GmrSD restriction endonucleases, C-terminal domain / Domain of unknown function DUF262 / GmrSD restriction endonuclease, N-terminal domain / DUF262 domain-containing protein
Function and homology information
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.28 Å
AuthorsZhou, Y.F. / Zhang, K.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: mBio / Year: 2026
Title: SspE-mediated immune defense: GTP hydrolysis as an allosteric switch coupling phosphorothioate recognition to DNA cleavage.
Authors: Yufeng Zhou / Kuo Zhang / Yu He / Haiyan Gao / Yuhang Zhong / Xiaoguang Wang / Meiying Wang / Lianrong Wang / Shi Chen /
Abstract: DNA phosphorothioate (PT) modification is an epigenetic mark that enables bacteria to discriminate self from non-self DNA, directing restriction effectors to cleave unmodified foreign DNA. In the PT- ...DNA phosphorothioate (PT) modification is an epigenetic mark that enables bacteria to discriminate self from non-self DNA, directing restriction effectors to cleave unmodified foreign DNA. In the PT-dependent Ssp system, SspE acts as the restriction effector that recognizes PT marks to block phage propagation. While the mechanism of the Streptomyces homolog (StSspE) is known, the basis for the exceptional potency of () 3234/A SspE (EcSspE) remained unclear. Here, we combine cryo-electron microscopy (cryo-EM), biochemistry, and functional assays to define its mechanism. The cryo-EM structure reveals that EcSspE forms a dynamic homotetramer with a side-by-side assembly, featuring a substantially reduced inter-subunit interface compared to the intertwined StSspE tetramer. A hydrophobic cavity harboring Y63 specifically recognizes the 5'-CCA-3' PT motif. This recognition triggers GTP hydrolysis via the essential residue R133. Hydrolysis, in turn, drives an asymmetric allosteric rearrangement that licenses the flexible C-terminal HNH nuclease domain for DNA cleavage. Disrupting PT sensing (Y63A), GTP hydrolysis (R133A), or nuclease activity (N724A) completely abolishes anti-phage defense, confirming strict functional coupling. Our work establishes a conserved "recognize-hydrolyze-activate" paradigm for SspE proteins, wherein PT-stimulated GTPase activity licenses the nuclease via an allosteric switch. The distinct tetrameric architecture of EcSspE likely underlies its enhanced activity by facilitating conformational dynamics. This study elucidates the precise molecular logic of a potent bacterial immune system and provides a framework for engineering phage resistance.IMPORTANCEBacterial antiphage defense systems must precisely destroy invaders while avoiding self-harm. This study provides a high-resolution molecular blueprint of the exceptionally potent PT-dependent Ssp system from 3234/A. We elucidate its conserved "recognize-hydrolyze-activate" mechanism: the effector EcSspE integrates PT recognition, GTP hydrolysis, and allosteric signaling to license DNA cleavage. Beyond this paradigm, we reveal that subtle evolutionary refinements in its quaternary architecture-a streamlined, side-by-side assembly with a reduced interface-amplify defensive output by enhancing conformational dynamics. This insight bridges structural biophysics and immunity. The system's strict PT-dependence ensures biosafety, and its defined mechanistic logic and key molecular switches (Y63, R133, N724) establish a framework for engineering programmable phage resistance, advancing both our understanding of host-virus conflict and our ability to harness it.
History
DepositionDec 3, 2025Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Apr 22, 2026Provider: repository / Type: Initial release
Revision 1.0Apr 22, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 22, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.0Apr 22, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Apr 22, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1May 27, 2026Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Revision 2.0May 27, 2026Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / EM metadata / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DUF262 domain-containing protein
B: DUF262 domain-containing protein
C: DUF262 domain-containing protein
D: DUF262 domain-containing protein


Theoretical massNumber of molelcules
Total (without water)376,5424
Polymers376,5424
Non-polymers00
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: Protein
DUF262 domain-containing protein


Mass: 94135.555 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: HV209_22330 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A8H9XTE3
Has protein modificationY

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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: SspE / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
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: 2700 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 48 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.20.1_4487model refinement
13cryoSPARC3D reconstruction
CTF correctionType: NONE
3D reconstructionResolution: 3.28 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 216504 / Symmetry type: POINT
RefinementHighest resolution: 3.28 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00313286
ELECTRON MICROSCOPYf_angle_d0.46917895
ELECTRON MICROSCOPYf_dihedral_angle_d3.9741737
ELECTRON MICROSCOPYf_chiral_restr0.041963
ELECTRON MICROSCOPYf_plane_restr0.0042265

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