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- EMDB-11172: Disulfide-locked early prepore intermedilysin-CD59 -

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Basic information

Entry
Database: EMDB / ID: EMD-11172
TitleDisulfide-locked early prepore intermedilysin-CD59
Map dataHalf map 1 of ILY-CD59 early prepore oligomer formed on a lipid nanodsic, 5 subunits
Sample
  • Complex: Early prepore of intermedilysin-CD59
    • Complex: Thiol-activated cytolysinCholesterol-dependent cytolysin
      • Protein or peptide: Thiol-activated cytolysinCholesterol-dependent cytolysin
    • Complex: CD59 glycoprotein
      • Protein or peptide: CD59 glycoprotein
Keywordsearly prepore / membrane-bound / oligomer / TOXIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsShah NR / Bubeck D
Funding support United Kingdom, 1 items
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, 2020-
Header (metadata) releaseNov 18, 2020-
Map releaseNov 18, 2020-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6zd0
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6zd0
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11172.png

Downloads & links

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Map

FileDownload / File: emd_11172.map.gz / Format: CCP4 / Size: 12.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHalf map 1 of ILY-CD59 early prepore oligomer formed on a lipid nanodsic, 5 subunits
Voxel sizeX=Y=Z: 1.4 Å
Density
Contour LevelBy AUTHOR: 0.022 / Movie #1: 0.04
Minimum - Maximum-0.072169185 - 0.19049014
Average (Standard dev.)0.0015517144 (±0.013816901)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions150150150
Spacing150150150
CellA=B=C: 210.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.41.41.4
M x/y/z150150150
origin x/y/z0.0000.0000.000
length x/y/z210.000210.000210.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS150150150
D min/max/mean-0.0720.1900.002

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Supplemental data

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Half map: Half map 2 of ILY-CD59 early prepore oligomer...

Fileemd_11172_half_map_1.map
AnnotationHalf map 2 of ILY-CD59 early prepore oligomer formed on a lipid nanodsic, 5 subunits
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 of ILY-CD59 early prepore oligomer...

Fileemd_11172_half_map_2.map
AnnotationHalf map 2 of ILY-CD59 early prepore oligomer formed on a lipid nanodsic, 5 subunits
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Early prepore of intermedilysin-CD59

EntireName: Early prepore of intermedilysin-CD59
Components
  • Complex: Early prepore of intermedilysin-CD59
    • Complex: Thiol-activated cytolysinCholesterol-dependent cytolysin
      • Protein or peptide: Thiol-activated cytolysinCholesterol-dependent cytolysin
    • Complex: CD59 glycoprotein
      • Protein or peptide: CD59 glycoprotein

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Supramolecule #1: Early prepore of intermedilysin-CD59

SupramoleculeName: Early prepore of intermedilysin-CD59 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Prepore is formed on a lipid bilayer on a nanodisc

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Supramolecule #2: Thiol-activated cytolysin

SupramoleculeName: Thiol-activated cytolysin / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Streptococcus intermedius (bacteria)

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Supramolecule #3: CD59 glycoprotein

SupramoleculeName: CD59 glycoprotein / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Thiol-activated cytolysin

MacromoleculeName: Thiol-activated cytolysin / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus intermedius (bacteria)
Molecular weightTheoretical: 59.100953 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGGSHHHHHH GMASMTGGQQ MGRDLYDDDD KDRWGSETPT KPKAAQTEKK TEKKPENSNS EAAKKALNDY IWGLQYDKLN ILTHQGEKL KNHSSREAFH RPGEYVVCEK KKQSISNATS KLSVSSANDD RIFPGALLKA DQSLLENLPT LIPVNRGKTT I SVNLPGLK ...String:
MGGSHHHHHH GMASMTGGQQ MGRDLYDDDD KDRWGSETPT KPKAAQTEKK TEKKPENSNS EAAKKALNDY IWGLQYDKLN ILTHQGEKL KNHSSREAFH RPGEYVVCEK KKQSISNATS KLSVSSANDD RIFPGALLKA DQSLLENLPT LIPVNRGKTT I SVNLPGLK NGESNLTVEN PSNSTVRTAV NNLVEKWIQN YSKTHAVPAR MQYESISAQS MSQLQAKFGA DFSKVGAPLN VD FSSVHKC EKQVFIANFR QVYYTASVDS PNSPSALFGS GITPTDLINR GVNSKTPPVY VSNVSYGRAM YVKFETTSKS TKV QAAIDA VVKGAKLKAG TEYENILKNT KITAVVLGGN PGEASKVITG NIDTLKDLIQ KGSNFSAQSP AVPISYTTSF VKDN SIATI QNNTDYIETK VTSYKDGALT LNHDGAFVAR FYVYWEELGH DADGYETIRS RSWSGNGYNR GAHYSTTLRF KGNVR NIRV KVLGATGLAW EPWRLIYSKN DLPLVPQRNI STWGTTLHPQ FEDKVVKDNT D

UniProtKB: Thiol-activated cytolysin

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Macromolecule #2: CD59 glycoprotein

MacromoleculeName: CD59 glycoprotein / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 9.204445 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MLQCYNCPNP TADCKTAVNC SSDFDACLIT KAGLQVYNKC WKFEHCNFND VTTRLRENEL TYYCCKKDLC NFNEQLENC

UniProtKB: CD59 glycoprotein

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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: 7.5
Component:
ConcentrationFormulaName
20.0 mM(HOCH2)3CNH2Tris
200.0 mMNaClSodium chloridesodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
Details: Grids were glow discharged before application of graphene oxide, then left to dry for 1 hour before use.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 294 K / Instrument: FEI VITROBOT MARK III / Details: Wait time of 60 s, blot time 2.5 s, blot force 3.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.1 µm / Nominal defocus min: 1.9000000000000001 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsPixel size 1.4 A
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Average exposure time: 1.0 sec. / Average electron dose: 66.0 e/Å2
Details: Some images were collected at a 30 degree tilt. Images were collected in movie mode at 39 frames per second.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
In silico model: A previous data set was used to generate a 8.6 A reconstruction, which was used as the startup model.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationNumber classes: 4 / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionNumber classes used: 2 / Resolution.type: BY AUTHOR / Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 51041

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Atomic model buiding 1

Initial modelPDB ID:

4bik


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsModel 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.
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

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

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