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- EMDB-40570: Structure of a bacterial gasdermin slinky-like oligomer -

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Basic information

Entry
Database: EMDB / ID: EMD-40570
TitleStructure of a bacterial gasdermin slinky-like oligomer
Map dataEM map of Vitiosangium bGSDM slinky oligomer
Sample
  • Complex: Vitiosangium bGSDM in an active slinky-like oligomeric conformation
    • Protein or peptide: Gasdermin bGSDM
Keywordsviral mimicry / gasdermin / caspase / autoinhibition / pyroptosis / bats / immunity / cell death / IMMUNE SYSTEM
Function / homologydefense response to virus / plasma membrane / cytoplasm / Gasdermin bGSDM
Function and homology information
Biological speciesVitiosangium sp. GDMCC 1.1324 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsJohnson AG / Mayer ML / Kranzusch PJ
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1DP2GM146250-01 United States
CitationJournal: bioRxiv / Year: 2023
Title: Structure and assembly of a bacterial gasdermin pore.
Authors: Alex G Johnson / Megan L Mayer / Stefan L Schaefer / Nora K McNamara-Bordewick / Gerhard Hummer / Philip J Kranzusch /
Abstract: In response to pathogen infection, gasdermin (GSDM) proteins form membrane pores that induce a host cell death process called pyroptosis. Studies of human and mouse GSDM pores reveal the functions ...In response to pathogen infection, gasdermin (GSDM) proteins form membrane pores that induce a host cell death process called pyroptosis. Studies of human and mouse GSDM pores reveal the functions and architectures of 24-33 protomers assemblies, but the mechanism and evolutionary origin of membrane targeting and GSDM pore formation remain unknown. Here we determine a structure of a bacterial GSDM (bGSDM) pore and define a conserved mechanism of pore assembly. Engineering a panel of bGSDMs for site-specific proteolytic activation, we demonstrate that diverse bGSDMs form distinct pore sizes that range from smaller mammalian-like assemblies to exceptionally large pores containing >50 protomers. We determine a 3.3 Å cryo-EM structure of a bGSDM in an active slinky-like oligomeric conformation and analyze bGSDM pores in a native lipid environment to create an atomic-level model of a full 52-mer bGSDM pore. Combining our structural analysis with molecular dynamics simulations and cellular assays, our results support a stepwise model of GSDM pore assembly and suggest that a covalently bound palmitoyl can leave a hydrophobic sheath and insert into the membrane before formation of the membrane-spanning β-strand regions. These results reveal the diversity of GSDM pores found in nature and explain the function of an ancient post-translational modification in enabling programmed host cell death.
History
DepositionApr 20, 2023-
Header (metadata) releaseMay 17, 2023-
Map releaseMay 17, 2023-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40570.png

Downloads & links

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Map

FileDownload / File: emd_40570.map.gz / Format: CCP4 / Size: 1000 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEM map of Vitiosangium bGSDM slinky oligomer
Voxel sizeX=Y=Z: 1.431 Å
Density
Contour LevelBy AUTHOR: 0.55
Minimum - Maximum-0.6345574 - 2.1764758
Average (Standard dev.)0.00005362098 (±0.034086373)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions640640640
Spacing640640640
CellA=B=C: 915.83997 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: EM half map A of Vitiosangium bGSDM slinky oligomer

Fileemd_40570_half_map_1.map
AnnotationEM half map A of Vitiosangium bGSDM slinky oligomer
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: EM half map B of Vitiosangium bGSDM slinky oligomer

Fileemd_40570_half_map_2.map
AnnotationEM half map B of Vitiosangium bGSDM slinky oligomer
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Vitiosangium bGSDM in an active slinky-like oligomeric conformation

EntireName: Vitiosangium bGSDM in an active slinky-like oligomeric conformation
Components
  • Complex: Vitiosangium bGSDM in an active slinky-like oligomeric conformation
    • Protein or peptide: Gasdermin bGSDM

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Supramolecule #1: Vitiosangium bGSDM in an active slinky-like oligomeric conformation

SupramoleculeName: Vitiosangium bGSDM in an active slinky-like oligomeric conformation
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Vitiosangium sp. GDMCC 1.1324 (bacteria)

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Macromolecule #1: Gasdermin bGSDM

MacromoleculeName: Gasdermin bGSDM / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Vitiosangium sp. GDMCC 1.1324 (bacteria)
Molecular weightTheoretical: 25.566246 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: SGLCSDPAIT YLKRLGYNVV RLPREGIQPL HLLGQQRGTV EYLGSLEKLI TQPPSEPPAI TRDQAAAGIN GQKTENLSFS IGINILKSV LAQFGAGAGI EAQYNQARKV RFEFSNVLAD SVEPLAVGQF LKMAEVDADN PVLKQYVLGN GRLYVITQVI K SNEFTVAA ...String:
SGLCSDPAIT YLKRLGYNVV RLPREGIQPL HLLGQQRGTV EYLGSLEKLI TQPPSEPPAI TRDQAAAGIN GQKTENLSFS IGINILKSV LAQFGAGAGI EAQYNQARKV RFEFSNVLAD SVEPLAVGQF LKMAEVDADN PVLKQYVLGN GRLYVITQVI K SNEFTVAA EKSGGGSIQL DVPEIQKVVG GKLKVEASVS SQSTVTYKGE KQLVFGFKCF EIGVKNGEIT LFASQLVPR

UniProtKB: Gasdermin bGSDM

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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

Concentration1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloridesodium chloride
20.0 mMHEPES-KOH buffer (pH 7.5)
5.16 mMN-Dodecyl-N,N-(dimethylammonio)butyrate

Details: 150 mM NaCl, 20 mM HEPES-HOH (pH 7.5), 5.15 mM DDMAB
GridModel: Quantifoil / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 27.0 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
DetailsThe sample was monodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.7000000000000001 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 8156 / Average electron dose: 51.8 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7n51

Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 132403
FSC plot (resolution estimation)

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