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- EMDB-63221: Cryo-EM structure of TIR-STING/c-di-GMP complex -

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Basic information

Entry
Database: EMDB / ID: EMD-63221
TitleCryo-EM structure of TIR-STING/c-di-GMP complex
Map data
Sample
  • Complex: TIR-STING/c-di-GMP complex
    • Protein or peptide: CD-NTase-associated protein 12
  • Ligand: 9,9'-[(2R,3R,3aS,5S,7aR,9R,10R,10aS,12S,14aR)-3,5,10,12-tetrahydroxy-5,12-dioxidooctahydro-2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9,2,8]tetraoxadiphosphacyclododecine-2,9-diyl]bis(2-amino-1,9-dihydro-6H-purin-6-one)
  • Ligand: CALCIUM ION
  • Ligand: water
KeywordsNADase / HYDROLASE
Function / homologyCD-NTase-associated protein 12/Pycsar effector protein, TIR domain / CAP12/Pycsar effector protein, TIR domain / Prokaryotic STING domain / Prokaryotic STING domain / NAD+ glycohydrolase / NADP+ nucleosidase activity / defense response to virus / nucleotide binding / CD-NTase-associated protein 12
Function and homology information
Biological speciesEpilithonimonas lactis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.88 Å
AuthorsLu DF / Liu S
Funding support China, 1 items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China) China
CitationJournal: mBio / Year: 2025
Title: Structural insights into distinct filamentation states reveal a regulatory mechanism for bacterial STING activation.
Authors: Yuchao Yang / Yueyue Liu / Xue Ma / Xuan Zhao / Jian Cao / Yu Liu / Shanqin Li / Jing Wu / Yuanzhu Gao / Lianwan Chen / Changxin Wu / Guijun Shang / Sheng Liu / Defen Lu /
Abstract: The cyclic oligonucleotide-based antiphage signaling system (CBASS) is a bacterial immune mechanism that was evolutionarily linked to the eukaryotic cGAS-STING pathway, which protects against phage ...The cyclic oligonucleotide-based antiphage signaling system (CBASS) is a bacterial immune mechanism that was evolutionarily linked to the eukaryotic cGAS-STING pathway, which protects against phage infection through abortive cell death. CBASS operons encode cyclic dinucleotide synthases (CD-NTases) and effector proteins (Caps), such as bacterial STING, which senses cyclic dinucleotides like 3'3'-c-di-GMP to trigger defense. Although bacterial STING oligomerizes into filaments upon ligand binding, the functional roles of distinct filament states remain unclear. Here, we resolve cryo-EM structures of TIR-STING (STING) bound to 3'3'-c-di-GMP, revealing two oligomeric states: spiral-shaped single filaments and fiber bundles composed of straight protofibrils. In spiral filaments, the STING domain sequesters the TIR domain's BB loop within a hydrophobic core, suppressing NADase activity. This inactive conformation is stabilized by interactions between the CBDα4 helix and the TIR domain, as well as a calcium-binding site. Conversely, fiber bundle formation-driven by inter-protofibril TIR domain interactions-disrupts these autoinhibitory contacts, liberating the BB loop to enable head-to-tail assembly of adjacent TIR domains into a composite NADase-active site. Calcium ions promote spiral filament assembly while inhibiting fiber bundles, revealing a dual regulatory role in tuning STING activation. Strikingly, this mechanism diverges from single-filament systems like STING, underscoring evolutionary diversity in STING signaling. Our findings establish distinct filament architectures as structural checkpoints governing bacterial STING activation, providing mechanistic insights into how conformational plasticity and environmental cues like calcium regulate abortive infection. These results highlight parallels between prokaryotic and eukaryotic immune strategies, emphasizing conserved principles in pathogen defense across domains of life.IMPORTANCEBacteria employ a sophisticated immune system, CBASS, evolutionarily related to human antiviral pathways, to defend against viral (phage) attacks. This study reveals how the bacterial protein STING acts as a molecular switch, transitioning between an inactive spiral structure stabilized by calcium ions and an active fiber bundle. When calcium levels drop, STING reorganizes into fiber bundles, activating its ability to degrade essential cellular molecules. This self-destructive mechanism halts phage replication by sacrificing the infected cell, protecting the bacterial population. The findings demonstrate how structural rearrangements govern life-or-death immune decisions, mirroring principles in human STING signaling. By uncovering calcium's role in regulating this process, the work deepens our understanding of microbial immunity and highlights shared strategies across domains of life. These insights could inspire novel antimicrobial therapies or bioengineered systems to combat infections, bridging fundamental science with practical applications in health and biotechnology.
History
DepositionJan 19, 2025-
Header (metadata) releaseAug 27, 2025-
Map releaseAug 27, 2025-
UpdateAug 27, 2025-
Current statusAug 27, 2025Processing site: PDBc / Status: Released

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Structure visualization

Downloads & links

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Map

FileDownload / File: emd_63221.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-1.5614777 - 3.3597307
Average (Standard dev.)0.005008072 (±0.09301798)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 212.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : TIR-STING/c-di-GMP complex

EntireName: TIR-STING/c-di-GMP complex
Components
  • Complex: TIR-STING/c-di-GMP complex
    • Protein or peptide: CD-NTase-associated protein 12
  • Ligand: 9,9'-[(2R,3R,3aS,5S,7aR,9R,10R,10aS,12S,14aR)-3,5,10,12-tetrahydroxy-5,12-dioxidooctahydro-2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9,2,8]tetraoxadiphosphacyclododecine-2,9-diyl]bis(2-amino-1,9-dihydro-6H-purin-6-one)
  • Ligand: CALCIUM ION
  • Ligand: water

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Supramolecule #1: TIR-STING/c-di-GMP complex

SupramoleculeName: TIR-STING/c-di-GMP complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Epilithonimonas lactis (bacteria)

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Macromolecule #1: CD-NTase-associated protein 12

MacromoleculeName: CD-NTase-associated protein 12 / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO / EC number: NAD+ glycohydrolase
Source (natural)Organism: Epilithonimonas lactis (bacteria)
Molecular weightTheoretical: 35.717707 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MRTRIFIGSS KEGLKEANYV KSRLEKANFE VFIWNDDIFK PNKNTLETLL NVASLFDFGI MIATKDDFTA SRDDIFETVR DNVVFEFGL FLGRLGENRA FALQENGAKL PSDLLGITIP KFEKTDDYFS NYNLNTEIDN IIKIINEKIS LGELGLLPST V LAIGYYEN ...String:
MRTRIFIGSS KEGLKEANYV KSRLEKANFE VFIWNDDIFK PNKNTLETLL NVASLFDFGI MIATKDDFTA SRDDIFETVR DNVVFEFGL FLGRLGENRA FALQENGAKL PSDLLGITIP KFEKTDDYFS NYNLNTEIDN IIKIINEKIS LGELGLLPST V LAIGYYEN FVSTVCDALH SLPTIKLNGI EYKDFVFNII IPNDLDADIK RRAQIYFKKM DIHEVKIDTN GRSFPLYLQI DE ENSGDVA VLYDMPTTLG GIDKAIEMYM KKGHIGKTSQ QQLLEERELR NFKTTLINLI NNNSFTKTFV KVIEE

UniProtKB: CD-NTase-associated protein 12

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Macromolecule #2: 9,9'-[(2R,3R,3aS,5S,7aR,9R,10R,10aS,12S,14aR)-3,5,10,12-tetrahydr...

MacromoleculeName: 9,9'-[(2R,3R,3aS,5S,7aR,9R,10R,10aS,12S,14aR)-3,5,10,12-tetrahydroxy-5,12-dioxidooctahydro-2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9,2,8]tetraoxadiphosphacyclododecine-2,9-diyl]bis(2-amino-1,9-dihydro-6H-purin-6-one)
type: ligand / ID: 2 / Number of copies: 4 / Formula: C2E
Molecular weightTheoretical: 690.411 Da
Chemical component information

ChemComp-C2E:
9,9'-[(2R,3R,3aS,5S,7aR,9R,10R,10aS,12S,14aR)-3,5,10,12-tetrahydroxy-5,12-dioxidooctahydro-2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9,2,8]tetraoxadiphosphacyclododecine-2,9-diyl]bis(2-amino-1,9-dihydro-6H-purin-6-one)

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Macromolecule #3: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 3 / Number of copies: 4 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 8 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.88 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 523122
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: ANGULAR RECONSTITUTION

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