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- PDB-6zd0: Disulfide-locked early prepore intermedilysin-CD59 -

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Basic information

Entry
Database: PDB / ID: 6zd0
TitleDisulfide-locked early prepore intermedilysin-CD59
Components
  • CD59 glycoprotein
  • Thiol-activated cytolysinCholesterol-dependent cytolysin
KeywordsTOXIN / early prepore / membrane-bound / oligomer
Function / homology
Function and homology information


negative regulation of activation of membrane attack complex / complement binding / regulation of complement-dependent cytotoxicity / regulation of complement activation / Cargo concentration in the ER / COPII-mediated vesicle transport / cholesterol binding / tertiary granule membrane / specific granule membrane / COPI-mediated anterograde transport ...negative regulation of activation of membrane attack complex / complement binding / regulation of complement-dependent cytotoxicity / regulation of complement activation / Cargo concentration in the ER / COPII-mediated vesicle transport / cholesterol binding / tertiary granule membrane / specific granule membrane / COPI-mediated anterograde transport / transport vesicle / endoplasmic reticulum-Golgi intermediate compartment membrane / Regulation of Complement cascade / ER to Golgi transport vesicle membrane / blood coagulation / toxin activity / killing of cells of another organism / vesicle / cell surface receptor signaling pathway / external side of plasma membrane / Golgi membrane / focal adhesion / Neutrophil degranulation / endoplasmic reticulum membrane / host cell plasma membrane / cell surface / extracellular space / extracellular exosome / extracellular region / membrane / metal ion binding / plasma membrane
Similarity search - Function
CD59 antigen, conserved site / Ly-6 / u-PAR domain signature. / Ly-6 antigen / uPA receptor -like domain / Thiol-activated cytolysin C-terminal / Thiol-activated cytolysin, C-terminal domain superfamily / Thiol-activated cytolysin beta sandwich domain / Thiol-activated cytolysin / Thiol-activated cytolysin superfamily / Thiol-activated cytolysin, alpha-beta domain superfamily / Thiol-activated cytolysin ...CD59 antigen, conserved site / Ly-6 / u-PAR domain signature. / Ly-6 antigen / uPA receptor -like domain / Thiol-activated cytolysin C-terminal / Thiol-activated cytolysin, C-terminal domain superfamily / Thiol-activated cytolysin beta sandwich domain / Thiol-activated cytolysin / Thiol-activated cytolysin superfamily / Thiol-activated cytolysin, alpha-beta domain superfamily / Thiol-activated cytolysin / u-PAR/Ly-6 domain / Ly-6 antigen/uPA receptor-like / Snake toxin-like superfamily
Similarity search - Domain/homology
CD59 glycoprotein / Thiol-activated cytolysin
Similarity search - Component
Biological speciesStreptococcus intermedius (bacteria)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsShah, N.R. / Bubeck, D.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Cancer Research UKC26409/A16099 United Kingdom
CitationJournal: Nat Commun / Year: 2020
Title: Structural basis for tuning activity and membrane specificity of bacterial cytolysins.
Authors: Nita R Shah / Tomas B Voisin / Edward S Parsons / Courtney M Boyd / Bart W Hoogenboom / Doryen Bubeck /
Abstract: Cholesterol-dependent cytolysins (CDCs) are pore-forming proteins that serve as major virulence factors for pathogenic bacteria. They target eukaryotic cells using different mechanisms, but all ...Cholesterol-dependent cytolysins (CDCs) are pore-forming proteins that serve as major virulence factors for pathogenic bacteria. They target eukaryotic cells using different mechanisms, but all require the presence of cholesterol to pierce lipid bilayers. How CDCs use cholesterol to selectively lyse cells is essential for understanding virulence strategies of several pathogenic bacteria, and for repurposing CDCs to kill new cellular targets. Here we address that question by trapping an early state of pore formation for the CDC intermedilysin, bound to the human immune receptor CD59 in a nanodisc model membrane. Our cryo electron microscopy map reveals structural transitions required for oligomerization, which include the lateral movement of a key amphipathic helix. We demonstrate that the charge of this helix is crucial for tuning lytic activity of CDCs. Furthermore, we discover modifications that overcome the requirement of cholesterol for membrane rupture, which may facilitate engineering the target-cell specificity of pore-forming proteins.
History
DepositionJun 13, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 18, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 25, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2May 1, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-11172
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  • Superimposition on EM map
  • EMDB-11172
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Thiol-activated cytolysin
B: CD59 glycoprotein
C: Thiol-activated cytolysin
D: CD59 glycoprotein
E: Thiol-activated cytolysin
F: CD59 glycoprotein


Theoretical massNumber of molelcules
Total (without water)204,9166
Polymers204,9166
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy, This assembly has been confirmed on nanodiscs, lipid monolayers, and liposomes with negative stain microscopy.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Thiol-activated cytolysin / Cholesterol-dependent cytolysin


Mass: 59100.953 Da / Num. of mol.: 3 / Mutation: I104C G244C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus intermedius (bacteria) / Gene: ily / Plasmid: pTricHis / Details (production host): IPTG-inducable promoter / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9LCB8
#2: Protein CD59 glycoprotein / 1F5 antigen / 20 kDa homologous restriction factor / HRF20 / MAC-inhibitory protein / MAC-IP / ...1F5 antigen / 20 kDa homologous restriction factor / HRF20 / MAC-inhibitory protein / MAC-IP / MEM43 antigen / Membrane attack complex inhibition factor / MACIF / Membrane inhibitor of reactive lysis / MIRL / Protectin


Mass: 9204.445 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CD59, MIC11, MIN1, MIN2, MIN3, MSK21 / Production host: Escherichia coli (E. coli) / References: UniProt: P13987

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Early prepore of intermedilysin-CD59COMPLEXPrepore is formed on a lipid bilayer on a nanodiscall0RECOMBINANT
2Thiol-activated cytolysinCholesterol-dependent cytolysinCOMPLEX#11RECOMBINANT
3CD59 glycoproteinCOMPLEX#21RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Streptococcus intermedius (bacteria)1338
23Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli BL21(DE3) (bacteria)469008
23Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris(HOCH2)3CNH21
2200 mMsodium chlorideNaClSodium chloride1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Grids were glow discharged before application of graphene oxide, then left to dry for 1 hour before use.
Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 294 K / Details: Wait time of 60 s, blot time 2.5 s, blot force 3

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: Pixel size 1.4 A
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3100 nm / Nominal defocus min: 1900 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1 sec. / Electron dose: 66 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k)
Details: Some images were collected at a 30 degree tilt. Images were collected in movie mode at 39 frames per second.

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Processing

EM software
IDNameVersionCategory
2crYOLOparticle selection
3EPUimage acquisition
5CTFFIND4CTF correction
8PHENIXmodel fitting
9Cootmodel fitting
11RELIONinitial Euler assignment
12RELIONfinal Euler assignment
13RELIONclassification
14RELION3D reconstruction
15PHENIXmodel refinement
CTF correctionType: NONE
3D reconstructionResolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 51041 / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: Model of ILY-CD59 was fit and refined into the central subunit density. A combination of rigid body fitting and global minimization with secondary structure restraints was used to fit and ...Details: Model of ILY-CD59 was fit and refined into the central subunit density. A combination of rigid body fitting and global minimization with secondary structure restraints was used to fit and refine the central subunit model. Then the central subunit model was rigid body fit as one body into the neighbouring subunits to generate a 3 subunit oligomer model.
Atomic model buildingPDB-ID: 4BIK

4bik


Accession code: 4BIK / Source name: PDB / Type: experimental model

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