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- EMDB-45451: Cryo-EM structure of the EaCDCL pore -

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Basic information

Entry
Database: EMDB / ID: EMD-45451
TitleCryo-EM structure of the EaCDCL pore
Map dataSharpened map
Sample
  • Complex: EaCDCL pore embedded in POPC liposome
    • Protein or peptide: Thiol-activated cytolysin family protein
  • Ligand: CALCIUM ION
Keywordspore-forming toxin / cholesterol-dependent cytolysin like / Elizabethkingia anophelis / MACPF / complement / TOXIN
Function / homologyThiol-activated cytolysin / Thiol-activated cytolysin superfamily / Thiol-activated cytolysin, alpha-beta domain superfamily / Thiol-activated cytolysin / cholesterol binding / Prokaryotic membrane lipoprotein lipid attachment site profile. / metal ion binding / Thiol-activated cytolysin family protein
Function and homology information
Biological speciesElizabethkingia anophelis Ag1 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.87 Å
AuthorsJohnstone BA / Christie MP / Morton CM / Brown HG / Hanssen E / Parker MW
Funding support Australia, 3 items
OrganizationGrant numberCountry
Australian Research Council (ARC)DP160101874 Australia
Australian Research Council (ARC)DP200102871 Australia
National Health and Medical Research Council (NHMRC, Australia)APP1194263 Australia
CitationJournal: Sci Adv / Year: 2025
Title: Structural basis for the pore-forming activity of a complement-like toxin.
Authors: Bronte A Johnstone / Michelle P Christie / Riya Joseph / Craig J Morton / Hamish G Brown / Eric Hanssen / Tristan C Sanford / Hunter L Abrahamsen / Rodney K Tweten / Michael W Parker /
Abstract: Pore-forming proteins comprise a highly diverse group of proteins exemplified by the membrane attack complex/perforin (MACPF), cholesterol-dependent cytolysin (CDC), and gasdermin superfamilies, ...Pore-forming proteins comprise a highly diverse group of proteins exemplified by the membrane attack complex/perforin (MACPF), cholesterol-dependent cytolysin (CDC), and gasdermin superfamilies, which all form gigantic pores (>150 angstroms). A recently found family of pore-forming toxins, called CDC-like proteins (CDCLs), are wide-spread in gut microbes and are a prevalent means of antibacterial antagonism. However, the structural aspects of how CDCLs assemble a pore remain a mystery. Here, we report the crystal structure of a proteolytically activated CDCL and cryo-electron microscopy structures of a prepore-like intermediate and a transmembrane pore providing detailed snapshots across the entire pore-forming pathway. These studies reveal a sophisticated array of regulatory features to ensure productive pore formation, and, thus, CDCLs straddle the MACPF, CDC, and gasdermin lineages of the giant pore superfamilies.
History
DepositionJun 23, 2024-
Header (metadata) releaseApr 9, 2025-
Map releaseApr 9, 2025-
UpdateApr 9, 2025-
Current statusApr 9, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45451.png

Downloads & links

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Map

FileDownload / File: emd_45451.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.32 Å/pix.
x 512 pix.
= 675.84 Å		1.32 Å/pix.
x 512 pix.
= 675.84 Å		1.32 Å/pix.
x 512 pix.
= 675.84 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.32 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.86
Minimum - Maximum-4.796567 - 7.5394726
Average (Standard dev.)-0.0054648276 (±0.13202275)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 675.84 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_45451_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Raw, unsharpened map

Fileemd_45451_additional_1.map
AnnotationRaw, unsharpened map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_45451_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_45451_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : EaCDCL pore embedded in POPC liposome

EntireName: EaCDCL pore embedded in POPC liposome
Components
  • Complex: EaCDCL pore embedded in POPC liposome
    • Protein or peptide: Thiol-activated cytolysin family protein
  • Ligand: CALCIUM ION

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Supramolecule #1: EaCDCL pore embedded in POPC liposome

SupramoleculeName: EaCDCL pore embedded in POPC liposome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Elizabethkingia anophelis Ag1 (bacteria)

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Macromolecule #1: Thiol-activated cytolysin family protein

MacromoleculeName: Thiol-activated cytolysin family protein / type: protein_or_peptide / ID: 1 / Number of copies: 30 / Enantiomer: LEVO
Source (natural)Organism: Elizabethkingia anophelis Ag1 (bacteria)
Molecular weightTheoretical: 39.494047 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GSHMRQDSEV NPLQVQNSSK VLNPNVTLPA NNLLYDEFFV SKESKLIEDS RNNKRKTSKI ASLNPYASTK AVLTTTSSTL TSDQIVVTV PQKTFIGGVY NSTTLDNLDY TPISYPLDPI TVSYSFPSDF IVDTIERPSL SSMRASVFKA MRAANFSGEQ S LAFDYNIK ...String:
GSHMRQDSEV NPLQVQNSSK VLNPNVTLPA NNLLYDEFFV SKESKLIEDS RNNKRKTSKI ASLNPYASTK AVLTTTSSTL TSDQIVVTV PQKTFIGGVY NSTTLDNLDY TPISYPLDPI TVSYSFPSDF IVDTIERPSL SSMRASVFKA MRAANFSGEQ S LAFDYNIK QFSYYSELKI AFGSNVNIGK IFSIDISGSN NKIKRTTGVF AKFTQKNFTI DMDLPADGNI FKNNSDLALT NG KNPVYIS SVTYGRLGII SIESNASYNE VNFALKAALT AGIVNGSLNI DSNSKKILEE SDLSVYLVGG RGTDAVQVIK GFA GFSNFI VNGGQFTPEA PGVPIYFSAS HASDNSVYYT TFTIDK

UniProtKB: Thiol-activated cytolysin family protein

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Macromolecule #2: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 30 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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: 7.4 / Details: HBS pH 7.4
GridModel: Quantifoil R2/2 / Material: GOLD / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV
DetailsProteoliposome sample. Act-EaCDCLL + act-EaCDCLS (1:2 molar ratio) were added to liposomes to yield a final sample with a liposome concentration of 3.95 mM and a 1:500 protein:lipid molar ratio. Sample was incubated at 37 degrees for 15 - 20 min before applying to grids.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 15971 / 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 / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 64000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 559234
Startup modelType of model: OTHER / Details: Ab initio model
Final reconstructionApplied symmetry - Point group: C30 (30 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.87 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 122300
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8g32


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementProtocol: RIGID BODY FIT
Output model

PDB-9ccp:
Cryo-EM structure of the EaCDCL pore

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