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- EMDB-71644: CryoEM structure of filament of Bacillus subtilis TIR domain prot... -

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Basic information

Entry
Database: EMDB / ID: EMD-71644
TitleCryoEM structure of filament of Bacillus subtilis TIR domain protein SpbK
Map datamain EM map
Sample
  • Complex: TIR domain protein from Bacillus subtilis
    • Protein or peptide: SpbK
KeywordsTIR domain protein / antiphage defence / ANTIVIRAL PROTEIN
Function / homologyTIR domain / ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase / NAD+ nucleosidase activity, cyclic ADP-ribose generating / Toll - interleukin 1 - resistance / TIR domain profile. / Toll/interleukin-1 receptor homology (TIR) domain / Toll/interleukin-1 receptor homology (TIR) domain superfamily / signal transduction / Uncharacterized protein YddK
Function and homology information
Biological speciesBacillus subtilis (bacteria)
Methodhelical reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsMishra BP / Ve T
Funding support Australia, 2 items
OrganizationGrant numberCountry
Australian Research Council (ARC)FT200100572 Australia
National Health and Medical Research Council (NHMRC, Australia)1196590 Australia
CitationJournal: Nat Commun / Year: 2025
Title: Molecular characterisation of the Bacillus subtilis SpbK antiphage defence system.
Authors: Biswa P Mishra / Christian L Loyo / Yanyao Cai / Thomas Litfin / Gause Miraj / Lou Brillault / Veronika Masic / Tamim Mosaiab / Premraj Rajaratnam / Santosh Rudrawar / Weixi Gu / Bostjan ...Authors: Biswa P Mishra / Christian L Loyo / Yanyao Cai / Thomas Litfin / Gause Miraj / Lou Brillault / Veronika Masic / Tamim Mosaiab / Premraj Rajaratnam / Santosh Rudrawar / Weixi Gu / Bostjan Kobe / Joseph P Gerdt / Alan D Grossman / Yun Shi / Thomas Ve /
Abstract: Bacteria have a variety of mechanisms for limiting predation by phages. SpbK is a Toll/interleukin-1 receptor (TIR) domain-containing antiphage defence protein from Bacillus subtilis that provides ...Bacteria have a variety of mechanisms for limiting predation by phages. SpbK is a Toll/interleukin-1 receptor (TIR) domain-containing antiphage defence protein from Bacillus subtilis that provides protection against the temperate phage SPβ via abortive infection. Here we structurally characterise SpbK and its interaction with the SPβ protein YonE. We demonstrate that SpbK is an NADase that produces both ADP-ribose (ADPR) and canonical cyclic ADPR with a N1-glycosidic bond (cADPR, also referred to as N1-cADPR). Combining cryo-EM, in silico predictions, site-directed mutagenesis, and phage infection assays, we show that formation of two-stranded head-to-tail assemblies of SpbK TIR domains is required for both NADase activity and antiphage defence. We also demonstrate that YonE is a dodecameric portal protein that activates the NADase function of SpbK by facilitating TIR domain clustering. Collectively, our results provide insight into how bacterial TIR NADases recognise phage infection.
History
DepositionJul 9, 2025-
Header (metadata) releaseDec 31, 2025-
Map releaseDec 31, 2025-
UpdateJan 14, 2026-
Current statusJan 14, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_71644.png

Downloads & links

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Map

FileDownload / File: emd_71644.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmain EM map
Projections & slices

Image control

Size
Brightness
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AxesZ (Sec.)Y (Row.)X (Col.)
0.97 Å/pix.
x 400 pix.
= 389.12 Å		0.97 Å/pix.
x 400 pix.
= 389.12 Å		0.97 Å/pix.
x 400 pix.
= 389.12 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.9728 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.17
Minimum - Maximum-0.58789426 - 0.8800144
Average (Standard dev.)0.0014353188 (±0.040371705)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 389.12 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_71644_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: sharpened map

Fileemd_71644_additional_1.map
Annotationsharpened map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: half map A

Fileemd_71644_half_map_1.map
Annotationhalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B

Fileemd_71644_half_map_2.map
Annotationhalf map B
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : TIR domain protein from Bacillus subtilis

EntireName: TIR domain protein from Bacillus subtilis
Components
  • Complex: TIR domain protein from Bacillus subtilis
    • Protein or peptide: SpbK

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Supramolecule #1: TIR domain protein from Bacillus subtilis

SupramoleculeName: TIR domain protein from Bacillus subtilis / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Bacillus subtilis (bacteria)

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Macromolecule #1: SpbK

MacromoleculeName: SpbK / type: protein_or_peptide / ID: 1 / Number of copies: 10 / Enantiomer: LEVO / EC number: ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 34.07552 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MHHHHHHSSG VDLGTENLYF QSNAMDTLKK LKFEANGIIG VLLDYSREPV LTNIIDQFRT SLESNDIELI RYSLEQLKTW YARNRNEIY KNEFVFNEFE HHETENKIKK FLDELPVVDE TEKSSITHFS SDQNRELEKK IFISHSSKDK IVCNAFVELL E DIGVSSED ...String:
MHHHHHHSSG VDLGTENLYF QSNAMDTLKK LKFEANGIIG VLLDYSREPV LTNIIDQFRT SLESNDIELI RYSLEQLKTW YARNRNEIY KNEFVFNEFE HHETENKIKK FLDELPVVDE TEKSSITHFS SDQNRELEKK IFISHSSKDK IVCNAFVELL E DIGVSSED IIYTSSPYHG IPGDEDIFEY LKKHLFKGAY VFYMLSDNYY DSVYCLNEMG ATWVNSNNCS TFILPGFKGE IK GVIDKNK KAFSLEEPID LFNLKEKILR MYDLTLEDKK WERIKAKFNT KLK

UniProtKB: Uncharacterized protein YddK

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 8.9 Å
Applied symmetry - Helical parameters - Δ&Phi: 79.3 °
Applied symmetry - Helical parameters - Axial symmetry: D1 (2x1 fold dihedral)
Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 116455
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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