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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-11172 | |||||||||
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Title | Disulfide-locked early prepore intermedilysin-CD59 | |||||||||
![]() | Half map 1 of ILY-CD59 early prepore oligomer formed on a lipid nanodsic, 5 subunits | |||||||||
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![]() | early prepore / ![]() ![]() ![]() | |||||||||
Function / homology | ![]() negative regulation of activation of membrane attack complex / complement binding / ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() Similarity search - Function | |||||||||
Biological species | ![]() ![]() ![]() ![]() | |||||||||
Method | ![]() ![]() | |||||||||
![]() | Shah NR / Bubeck D | |||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 8.9 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 20.1 KB 20.1 KB | Display Display | ![]() |
Images | ![]() | 146 KB | ||
Filedesc metadata | ![]() | 6.5 KB | ||
Others | ![]() ![]() | 9.7 MB 9.7 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 6zd0MC M: atomic model generated by this map C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | Half map 1 of ILY-CD59 early prepore oligomer formed on a lipid nanodsic, 5 subunits | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.4 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Half map: Half map 2 of ILY-CD59 early prepore oligomer...
File | emd_11172_half_map_1.map | ||||||||||||
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Annotation | Half map 2 of ILY-CD59 early prepore oligomer formed on a lipid nanodsic, 5 subunits | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map 2 of ILY-CD59 early prepore oligomer...
File | emd_11172_half_map_2.map | ||||||||||||
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Annotation | Half map 2 of ILY-CD59 early prepore oligomer formed on a lipid nanodsic, 5 subunits | ||||||||||||
Projections & Slices |
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Density Histograms |
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Sample components
-Entire : Early prepore of intermedilysin-CD59
Entire | Name: Early prepore of intermedilysin-CD59 |
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Components |
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-Supramolecule #1: Early prepore of intermedilysin-CD59
Supramolecule | Name: 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
Supramolecule | Name: Thiol-activated cytolysin / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 |
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Source (natural) | Organism: ![]() ![]() |
-Supramolecule #3: CD59 glycoprotein
Supramolecule | Name: CD59 glycoprotein / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2 |
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Source (natural) | Organism: ![]() ![]() |
-Macromolecule #1: Thiol-activated cytolysin
Macromolecule | Name: Thiol-activated cytolysin / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 59.100953 KDa |
Recombinant expression | Organism: ![]() ![]() ![]() |
Sequence | String: 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: ![]() |
-Macromolecule #2: CD59 glycoprotein
Macromolecule | Name: CD59 glycoprotein / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 9.204445 KDa |
Recombinant expression | Organism: ![]() ![]() ![]() |
Sequence | String: MLQCYNCPNP TADCKTAVNC SSDFDACLIT KAGLQVYNKC WKFEHCNFND VTTRLRENEL TYYCCKKDLC NFNEQLENC UniProtKB: CD59 glycoprotein |
-Experimental details
-Structure determination
Method | ![]() |
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Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.5 Component:
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Grid | Model: 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. | |||||||||
Vitrification | Cryogen 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
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD![]() |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Details | Pixel size 1.4 A |
Image recording | Film 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 model | Type 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. |
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Initial angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: RELION |
Final 3D classification | Number classes: 4 / Software - Name: RELION |
Final angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: RELION |
Final reconstruction | Number 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 |
-Atomic model buiding 1
Initial model | PDB ID: Chain - Source name: PDB / Chain - Initial model type: experimental model |
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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. |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
Output model | ![]() PDB-6zd0: |