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- EMDB-67421: Cryo-EM structure of SspE-R133A from E.coli -

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Basic information

Entry
Database: EMDB / ID: EMD-67421
TitleCryo-EM structure of SspE-R133A from E.coli
Map data
Sample
  • Complex: SspE
    • Protein or peptide: DUF262 domain-containing protein
KeywordsNuclease / DNA phosphorothioate / defense system / DNA BINDING PROTEIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.81 Å
AuthorsZhou YF / Zhang K
Funding support1 items
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, 2025-
Header (metadata) releaseApr 22, 2026-
Map releaseApr 22, 2026-
UpdateMay 27, 2026-
Current statusMay 27, 2026Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_67421.png

Downloads & links

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Map

FileDownload / File: emd_67421.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesX (Sec.)Y (Row.)Z (Col.)
0.86 Å/pix.
x 420 pix.
= 359.1 Å		0.86 Å/pix.
x 420 pix.
= 359.1 Å		0.86 Å/pix.
x 420 pix.
= 359.1 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.855 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.0
Minimum - Maximum-26.891148000000001 - 54.054172999999999
Average (Standard dev.)-0.000000000000478 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 359.1 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_67421_half_map_1.map
Projections & Slices
AxesZYX

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Histogram

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Half map: #1

Fileemd_67421_half_map_2.map
Projections & Slices
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Sample components

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Entire : SspE

EntireName: SspE
Components
  • Complex: SspE
    • Protein or peptide: DUF262 domain-containing protein

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Supramolecule #1: SspE

SupramoleculeName: SspE / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: DUF262 domain-containing protein

MacromoleculeName: DUF262 domain-containing protein / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 94.049438 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MIIAKSSIVG KITFVVKCRN VNFLINSIKS RGYMEIKDVF GAQPKSVWEY LCENGQGLYV PAYQRQYSWD KPKITRLIED ICHGFTTLI SRDDAITFLG TIIAIHDTNL VTVDPIVKGD VPSRVMTIID GQQALTTLLL VNTVLHEEIK IRLVKKINKK S EADADIWL ...String:
MIIAKSSIVG KITFVVKCRN VNFLINSIKS RGYMEIKDVF GAQPKSVWEY LCENGQGLYV PAYQRQYSWD KPKITRLIED ICHGFTTLI SRDDAITFLG TIIAIHDTNL VTVDPIVKGD VPSRVMTIID GQQALTTLLL VNTVLHEEIK IRLVKKINKK S EADADIWL VEECMKVIGR LAKTFEEDKD YGDENFRYYP RMIRAYDDSW SRKKDKASYK SAIGHYLHTY GKYGREEIKK NF KYDPPES EQENSSKYKP LSEGRKTVYA LVKNICKSND STGKSSVKSD QLELELPEIS SILENEKFQN LLLKSEFPEY VKD KLIKND DQSFEELIRL ILFANFVLDR VAITIVTAKN EDYAFDMFES LNTTGEPLTA FETFKPKIIN AEKLSGYERS KSHQ YVEAI ENYLESTGKS NDKQEATSRL IVSFALAEKG EKLSKRLSEQ RRFLKDSFEK LPELKQQQEF VRHLSHAALF IRYSW PDDK SLTSSIYSAE EAQTDEVILC IDLLRKFNHT ITLGPLIRFY SEIRRVSPEF RTIAINNFID AVKAITAFSV LWRSSR RTT ENIDSHYRRL MMYGYEDVGL KPLARDMNEF GSEITLNVIG LKRAFLSILA KEGNVGSKDE WVKAISKIPA YSNQKEI TR FILLAAAHDS VEDKNIPGLT VAGREGTLPM LDIKNWKGEQ LQTIEHIAPQ EKSHEGWLED IYEEQELIDR LGNLTLLP S RENSSLKNGS WAKKKLFYKI LSAMTPDQLE PLQEQGKIQG INLAQSTSEL LSKSRYLPLV KAVALVEGDW TKELIEKRS VRIAELAWDR IHAWLNVE

UniProtKB: DUF262 domain-containing protein

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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: GATAN K3 (6k x 4k) / Average electron dose: 45.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.6 µm / Nominal defocus min: 0.7000000000000001 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: NONE
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.81 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 179737
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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