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

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Basic information

Entry
Database: PDB / ID: 9ccp
TitleCryo-EM structure of the EaCDCL pore
ComponentsThiol-activated cytolysin family protein
KeywordsTOXIN / pore-forming toxin / cholesterol-dependent cytolysin like / Elizabethkingia anophelis / MACPF / complement
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.87 Å
AuthorsJohnstone, B.A. / Christie, M.P. / Morton, C.M. / Brown, H.G. / Hanssen, E. / Parker, M.W.
Funding support Australia, 3items
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, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 9, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Thiol-activated cytolysin family protein
B: Thiol-activated cytolysin family protein
C: Thiol-activated cytolysin family protein
D: Thiol-activated cytolysin family protein
E: Thiol-activated cytolysin family protein
F: Thiol-activated cytolysin family protein
G: Thiol-activated cytolysin family protein
H: Thiol-activated cytolysin family protein
I: Thiol-activated cytolysin family protein
J: Thiol-activated cytolysin family protein
K: Thiol-activated cytolysin family protein
L: Thiol-activated cytolysin family protein
M: Thiol-activated cytolysin family protein
N: Thiol-activated cytolysin family protein
O: Thiol-activated cytolysin family protein
P: Thiol-activated cytolysin family protein
Q: Thiol-activated cytolysin family protein
R: Thiol-activated cytolysin family protein
S: Thiol-activated cytolysin family protein
T: Thiol-activated cytolysin family protein
U: Thiol-activated cytolysin family protein
V: Thiol-activated cytolysin family protein
W: Thiol-activated cytolysin family protein
X: Thiol-activated cytolysin family protein
Y: Thiol-activated cytolysin family protein
Z: Thiol-activated cytolysin family protein
AA: Thiol-activated cytolysin family protein
BA: Thiol-activated cytolysin family protein
CA: Thiol-activated cytolysin family protein
DA: Thiol-activated cytolysin family protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,186,02460
Polymers1,184,82130
Non-polymers1,20230
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ...
Thiol-activated cytolysin family protein / EaCDCL short


Mass: 39494.047 Da / Num. of mol.: 30
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Elizabethkingia anophelis Ag1 (bacteria)
Gene: JCR23_19030 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A7T7HCZ8
#2: Chemical...
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 30 / Source method: obtained synthetically / Formula: Ca
Has ligand of interestN
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: EaCDCL pore embedded in POPC liposome / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Elizabethkingia anophelis Ag1 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.4 / Details: HBS pH 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Proteoliposome 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 ...Details: Proteoliposome 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.
Specimen supportGrid material: GOLD / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 64000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 15971
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 559234
SymmetryPoint symmetry: C30 (30 fold cyclic)
3D reconstructionResolution: 2.87 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 122300 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 8G32

8g32


Pdb chain-ID: A / Accession code: 8G32 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00459250
ELECTRON MICROSCOPYf_angle_d0.63180520
ELECTRON MICROSCOPYf_dihedral_angle_d3.0448070
ELECTRON MICROSCOPYf_chiral_restr0.0459210
ELECTRON MICROSCOPYf_plane_restr0.00510440

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