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- EMDB-26618: SfSTING with cGAMP (masked) -

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Basic information

Entry
Database: EMDB / ID: EMD-26618
TitleSfSTING with cGAMP (masked)
Map dataSfSTING with cGAMP (masked) sharpened map
Sample
  • Complex: SfSTING with cGAMP
    • Protein or peptide: CD-NTase-associated protein 12
  • Ligand: 2-amino-9-[(2R,3R,3aS,5R,7aR,9R,10R,10aS,12R,14aR)-9-(6-amino-9H-purin-9-yl)-3,5,10,12-tetrahydroxy-5,12-dioxidooctahydro-2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9,2,8]tetraoxadiphosphacyclododecin-2-yl]-1,9-dihydro-6H-purin-6-one
KeywordsSTING / bacterial / filament / ANTIVIRAL PROTEIN
Function / homology
Function and homology information


NAD+ glycohydrolase / NADP+ nucleosidase activity / NAD+ 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
CD-NTase-associated protein 12
Similarity search - Component
Biological speciesSphingobacterium faecium (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsMorehouse BR / Yip MCJ / Keszei AFA / McNamara-Bordewick NK / Shao S / Kranzusch PJ
Funding support United States, 11 items
OrganizationGrant numberCountry
The Pew Charitable Trusts United States
Burroughs Wellcome Fund United States
The G. Harold and Leila Y. Mathers Foundation United States
The Mark Foundation United States
Parker Institute for Cancer Immunotherapy United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1DP2GM146250 United States
The Vallee Foundation United States
David and Lucile Packard Foundation United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1DP2GM137415 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F32GM133063 United States
American Heart Association287375208 United States
CitationJournal: Nature / Year: 2022
Title: Cryo-EM structure of an active bacterial TIR-STING filament complex.
Authors: Benjamin R Morehouse / Matthew C J Yip / Alexander F A Keszei / Nora K McNamara-Bordewick / Sichen Shao / Philip J Kranzusch /
Abstract: Stimulator of interferon genes (STING) is an antiviral signalling protein that is broadly conserved in both innate immunity in animals and phage defence in prokaryotes. Activation of STING requires ...Stimulator of interferon genes (STING) is an antiviral signalling protein that is broadly conserved in both innate immunity in animals and phage defence in prokaryotes. Activation of STING requires its assembly into an oligomeric filament structure through binding of a cyclic dinucleotide, but the molecular basis of STING filament assembly and extension remains unknown. Here we use cryogenic electron microscopy to determine the structure of the active Toll/interleukin-1 receptor (TIR)-STING filament complex from a Sphingobacterium faecium cyclic-oligonucleotide-based antiphage signalling system (CBASS) defence operon. Bacterial TIR-STING filament formation is driven by STING interfaces that become exposed on high-affinity recognition of the cognate cyclic dinucleotide signal c-di-GMP. Repeating dimeric STING units stack laterally head-to-head through surface interfaces, which are also essential for human STING tetramer formation and downstream immune signalling in mammals. The active bacterial TIR-STING structure reveals further cross-filament contacts that brace the assembly and coordinate packing of the associated TIR NADase effector domains at the base of the filament to drive NAD hydrolysis. STING interface and cross-filament contacts are essential for cell growth arrest in vivo and reveal a stepwise mechanism of activation whereby STING filament assembly is required for subsequent effector activation. Our results define the structural basis of STING filament formation in prokaryotic antiviral signalling.
History
DepositionApr 9, 2022-
Header (metadata) releaseJul 27, 2022-
Map releaseJul 27, 2022-
UpdateFeb 14, 2024-
Current statusFeb 14, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26618.png

Downloads & links

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Map

FileDownload / File: emd_26618.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSfSTING with cGAMP (masked) sharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 280 pix.
= 308. Å		1.1 Å/pix.
x 280 pix.
= 308. Å		1.1 Å/pix.
x 280 pix.
= 308. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.044029813 - 0.088274375
Average (Standard dev.)0.00019243006 (±0.002369902)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 308.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: SfSTING with cGAMP (masked) unsharpened map

Fileemd_26618_additional_1.map
AnnotationSfSTING with cGAMP (masked) unsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: SfSTING with cGAMP (masked) half map 1

Fileemd_26618_half_map_1.map
AnnotationSfSTING with cGAMP (masked) half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: SfSTING with cGAMP (masked) half map 2

Fileemd_26618_half_map_2.map
AnnotationSfSTING with cGAMP (masked) half map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : SfSTING with cGAMP

EntireName: SfSTING with cGAMP
Components
  • Complex: SfSTING with cGAMP
    • Protein or peptide: CD-NTase-associated protein 12
  • Ligand: 2-amino-9-[(2R,3R,3aS,5R,7aR,9R,10R,10aS,12R,14aR)-9-(6-amino-9H-purin-9-yl)-3,5,10,12-tetrahydroxy-5,12-dioxidooctahydro-2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9,2,8]tetraoxadiphosphacyclododecin-2-yl]-1,9-dihydro-6H-purin-6-one

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Supramolecule #1: SfSTING with cGAMP

SupramoleculeName: SfSTING with cGAMP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Sphingobacterium faecium (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: Sphingobacterium faecium (bacteria)
Molecular weightTheoretical: 36.956051 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MHHHHHHGSK KRIFIGSSSE QLTILNEIVD LLGDDVECIP WTDAFALNKS GLDSLIKQTR LADYSILIAT KDDLTKQRGE SLTKPRDNV VFEFGLFLGA AGPEKCYLIA EEDTDLPTDL DGITVAKFTR NSGQYNSLDK IVESIRTHLV KIAEMSQLGL L PSTALAIG ...String:
MHHHHHHGSK KRIFIGSSSE QLTILNEIVD LLGDDVECIP WTDAFALNKS GLDSLIKQTR LADYSILIAT KDDLTKQRGE SLTKPRDNV VFEFGLFLGA AGPEKCYLIA EEDTDLPTDL DGITVAKFTR NSGQYNSLDK IVESIRTHLV KIAEMSQLGL L PSTALAIG YYNSFIKRVC EEIHGSECVE LEGKKIKVKS FRVDVVIPET LDDNGVGNFT TLYNKRYGLS KATTCTNPAL LG TRGFPFH FKVDPPDANQ ESPVDIHLLD IPSTLSTIVE SLKLYLPSNQ VGQDFDMDYL EMRELENFAK VLKYLIGRNA ATK GYVNVL TNVKL

UniProtKB: CD-NTase-associated protein 12

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Macromolecule #2: 2-amino-9-[(2R,3R,3aS,5R,7aR,9R,10R,10aS,12R,14aR)-9-(6-amino-9H-...

MacromoleculeName: 2-amino-9-[(2R,3R,3aS,5R,7aR,9R,10R,10aS,12R,14aR)-9-(6-amino-9H-purin-9-yl)-3,5,10,12-tetrahydroxy-5,12-dioxidooctahydro-2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9,2,8]tetraoxadiphosphacyclododecin-2- ...Name: 2-amino-9-[(2R,3R,3aS,5R,7aR,9R,10R,10aS,12R,14aR)-9-(6-amino-9H-purin-9-yl)-3,5,10,12-tetrahydroxy-5,12-dioxidooctahydro-2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9,2,8]tetraoxadiphosphacyclododecin-2-yl]-1,9-dihydro-6H-purin-6-one
type: ligand / ID: 2 / Number of copies: 4 / Formula: 4BW
Molecular weightTheoretical: 674.411 Da
Chemical component information

ChemComp-4BW:
2-amino-9-[(2R,3R,3aS,5R,7aR,9R,10R,10aS,12R,14aR)-9-(6-amino-9H-purin-9-yl)-3,5,10,12-tetrahydroxy-5,12-dioxidooctahydro-2H,7H-difuro[3,2-d:3',2'-j][1,3,7,9,2,8]tetraoxadiphosphacyclododecin-2-yl]-1,9-dihydro-6H-purin-6-one

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 52.9 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.4000000000000001 µm
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 105567
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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