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- PDB-9lmq: Cryo-EM structure of TIR-STING/c-di-GMP complex -

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Basic information

Entry
Database: PDB / ID: 9lmq
TitleCryo-EM structure of TIR-STING/c-di-GMP complex
ComponentsCD-NTase-associated protein 12
KeywordsHYDROLASE / NADase
Function / homology
Function and homology information


NAD+ glycohydrolase / NADP+ nucleosidase activity / defense response to virus / nucleotide binding
Similarity search - Function
CD-NTase-associated protein 12/Pycsar effector protein, TIR domain / CAP12/Pycsar effector protein, TIR domain / Prokaryotic STING domain / Prokaryotic STING domain
Similarity search - Domain/homology
Chem-C2E / CD-NTase-associated protein 12
Similarity search - Component
Biological speciesEpilithonimonas lactis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.88 Å
AuthorsLu, D.F. / Liu, S.
Funding support China, 1items
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, 2025Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Aug 27, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CD-NTase-associated protein 12
B: CD-NTase-associated protein 12
G: CD-NTase-associated protein 12
I: CD-NTase-associated protein 12
H: CD-NTase-associated protein 12
J: CD-NTase-associated protein 12
C: CD-NTase-associated protein 12
D: CD-NTase-associated protein 12
hetero molecules


Theoretical massNumber of molelcules
Total (without water)288,66416
Polymers285,7428
Non-polymers2,9228
Water1448
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
CD-NTase-associated protein 12 / NAD(+) hydrolase / TIR-STING


Mass: 35717.707 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Epilithonimonas lactis (bacteria) / Gene: IO89_10965 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A085BE66, NAD+ glycohydrolase
#2: Chemical
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) / c-di-GMP / Cyclic diguanosine monophosphate


Mass: 690.411 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C20H24N10O14P2 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
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: TIR-STING/c-di-GMP complex / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Epilithonimonas lactis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
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: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.19.2_4158 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.88 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 523122 / Symmetry type: POINT
RefinementHighest resolution: 2.88 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00220565
ELECTRON MICROSCOPYf_angle_d0.4727787
ELECTRON MICROSCOPYf_dihedral_angle_d3.8692657
ELECTRON MICROSCOPYf_chiral_restr0.0413157
ELECTRON MICROSCOPYf_plane_restr0.0033541

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