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- EMDB-45448: Double-stacked pore and prepore-like complex (C1 symmetry) -

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Basic information

Entry
Database: EMDB / ID: EMD-45448
TitleDouble-stacked pore and prepore-like complex (C1 symmetry)
Map dataSharpened map
Sample
  • Complex: EaCDCL pore embedded in POPC liposome
    • Protein or peptide: EaCDCL short
    • Protein or peptide: EaCDCL long
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 / Hemolysin / Thiol-activated cytolysin family protein
Function and homology information
Biological speciesElizabethkingia anophelis Ag1 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.35 Å
AuthorsJohnstone BA / Christie MP / Morton CJ / 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_45448.png

Downloads & links

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Map

FileDownload / File: emd_45448.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: 0.49
Minimum - Maximum-2.2423263 - 4.7969613
Average (Standard dev.)-0.003971271 (±0.12882313)
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_45448_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_45448_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: #2

Fileemd_45448_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_45448_half_map_2.map
Projections & Slices
AxesZYX

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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: EaCDCL short
    • Protein or peptide: EaCDCL long

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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: all
Source (natural)Organism: Elizabethkingia anophelis Ag1 (bacteria)

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Macromolecule #1: EaCDCL short

MacromoleculeName: EaCDCL short / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Elizabethkingia anophelis Ag1 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GSHMRQDSEV NPLQVQNSSK VLNPNVTLPA NNLLYDEFFV SKESKLIEDS RNNKRKTSKI ASLNPYASTK AVLTTTSSTL TSDQIVVTVP QKTFIGGVYN STTLDNLDYT PISYPLDPIT VSYSFPSDFI VDTIERPSLS SMRASVFKAM RAANFSGEQS LAFDYNIKQF ...String:
GSHMRQDSEV NPLQVQNSSK VLNPNVTLPA NNLLYDEFFV SKESKLIEDS RNNKRKTSKI ASLNPYASTK AVLTTTSSTL TSDQIVVTVP QKTFIGGVYN STTLDNLDYT PISYPLDPIT VSYSFPSDFI VDTIERPSLS SMRASVFKAM RAANFSGEQS LAFDYNIKQF SYYSELKIAF GSNVNIGKIF SIDISGSNNK IKRTTGVFAK FTQKNFTIDM DLPADGNIFK NNSDLALTNG KNPVYISSVT YGRLGIISIE SNASYNEVNF ALKAALTAGI VNGSLNIDSN SKKILEESDL SVYLVGGRGT DAVQVIKGFA GFSNFIVNGG QFTPEAPGVP IYFSASHASD NSVYYTTFTI DK

UniProtKB: Thiol-activated cytolysin family protein

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Macromolecule #2: EaCDCL long

MacromoleculeName: EaCDCL long / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Elizabethkingia anophelis Ag1 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GSHMTDNLPD RSSVTNNMAR VELPVINITS FGTKPSFLNI QKSASTKSLN LIAENSGDTE TKEFESSESV VLNHLNRYVF PGSLLMGNSI QDLNYKPVFA SLNPITVSLS IPAINQNTAI TITNPSLSAT RAAVYNYLKT ADFTQNGQLS YSIQQFSSYD ELKVAFGSNV ...String:
GSHMTDNLPD RSSVTNNMAR VELPVINITS FGTKPSFLNI QKSASTKSLN LIAENSGDTE TKEFESSESV VLNHLNRYVF PGSLLMGNSI QDLNYKPVFA SLNPITVSLS IPAINQNTAI TITNPSLSAT RAAVYNYLKT ADFTQNGQLS YSIQQFSSYD ELKVAFGSNV NSRNLFGKNS SSTNVEEGMV ARQSGFYVKF YQTSFTLDMD VPNGSLVKDN NFDSEGIEPV YVSSISYGRM GILAIETNEK AEDAKRIINE TFNKLFYKKQ TNFSQEEKSF IEGADFNLYL VGGDGSTASQ SFKGYEAFVN HVSQGTFSKD QPGVPIFCSY SYLKDNSPVK TKFKFDIKRP PLYVKLVKEN MKDINFNDPD GGIYDNKKEA ILKIYFYKNR SLVPTLPNPY INFKIREKKK KWQSIAPVYY SSLDQVPFNI SERILTKQNT LQNIFATIQT QDNTEFSLIS RIIRGGGRQN PVPAGFRAIE INDYELVEDS NYIIIKD

UniProtKB: Hemolysin

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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
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: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.35 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.0) / Number images used: 123280
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.0)
FSC plot (resolution estimation)

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