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- PDB-8b0h: 2C9, C5b9-CD59 cryoEM structure -

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Basic information

Entry
Database: PDB / ID: 8b0h
Title2C9, C5b9-CD59 cryoEM structure
Components
  • (Complement component ...) x 6
  • CD59 glycoprotein
  • Complement C5Complement component 5
KeywordsMEMBRANE PROTEIN / Complement / CD59 / MAC
Function / homology
Function and homology information


negative regulation of activation of membrane attack complex / cell killing / Terminal pathway of complement / membrane attack complex / complement binding / regulation of complement-dependent cytotoxicity / other organism cell membrane / regulation of complement activation / Activation of C3 and C5 / negative regulation of macrophage chemotaxis ...negative regulation of activation of membrane attack complex / cell killing / Terminal pathway of complement / membrane attack complex / complement binding / regulation of complement-dependent cytotoxicity / other organism cell membrane / regulation of complement activation / Activation of C3 and C5 / negative regulation of macrophage chemotaxis / Cargo concentration in the ER / complement activation, alternative pathway / complement activation / chemokine activity / COPII-mediated vesicle transport / retinol binding / endopeptidase inhibitor activity / tertiary granule membrane / positive regulation of vascular endothelial growth factor production / specific granule membrane / COPI-mediated anterograde transport / complement activation, classical pathway / positive regulation of chemokine production / transport vesicle / endoplasmic reticulum-Golgi intermediate compartment membrane / Peptide ligand-binding receptors / Regulation of Complement cascade / protein homooligomerization / ER to Golgi transport vesicle membrane / extracellular vesicle / chemotaxis / positive regulation of immune response / blood coagulation / G alpha (i) signalling events / blood microparticle / killing of cells of another organism / in utero embryonic development / vesicle / cell surface receptor signaling pathway / immune response / inflammatory response / G protein-coupled receptor signaling pathway / external side of plasma membrane / Golgi membrane / signaling receptor binding / focal adhesion / innate immune response / Neutrophil degranulation / protein-containing complex binding / endoplasmic reticulum membrane / cell surface / extracellular space / extracellular exosome / extracellular region / membrane / plasma membrane
Similarity search - Function
Kazal-type serine protease inhibitor domain / : / : / Kazal-type serine protease inhibitor domain / Complement component C7, FIM2 N-terminal / Complement component C7, Kazal domain / : / Complement component C6, KAZAL domain / Complement component C8 gamma chain / : ...Kazal-type serine protease inhibitor domain / : / : / Kazal-type serine protease inhibitor domain / Complement component C7, FIM2 N-terminal / Complement component C7, Kazal domain / : / Complement component C6, KAZAL domain / Complement component C8 gamma chain / : / Complement components C8A/B/C6, EGF-like domain / CD59 antigen, conserved site / Ly-6 / u-PAR domain signature. / Membrane attack complex component/perforin/complement C9 / Ly-6 antigen / uPA receptor -like domain / Alpha-1-microglobulin / Factor I / membrane attack complex / factor I membrane attack complex / Membrane attack complex component/perforin domain, conserved site / Membrane attack complex/perforin (MACPF) domain signature. / u-PAR/Ly-6 domain / Ly-6 antigen/uPA receptor-like / : / Complement component 5, CUB domain / membrane-attack complex / perforin / Membrane attack complex/perforin (MACPF) domain profile. / MAC/Perforin domain / Membrane attack complex component/perforin (MACPF) domain / Complement C3/4/5, macroglobulin domain MG1 / Macroglobulin domain MG1 / Anaphylatoxin, complement system domain / Anaphylatoxin domain signature. / Anaphylatoxin/fibulin / Anaphylatoxin, complement system / Anaphylotoxin-like domain / Anaphylatoxin domain profile. / Anaphylatoxin homologous domain / Netrin C-terminal Domain / Netrin module, non-TIMP type / UNC-6/NTR/C345C module / Kazal domain / Kazal domain profile. / Netrin domain / NTR domain profile. / Alpha-macroglobulin, receptor-binding / Alpha-macroglobulin, receptor-binding domain superfamily / Macroglobulin domain MG4 / Macroglobulin domain MG3 / A-macroglobulin receptor binding domain / Macroglobulin domain MG4 / Macroglobulin domain MG3 / A-macroglobulin receptor / Tissue inhibitor of metalloproteinases-like, OB-fold / Alpha-2-macroglobulin / Macroglobulin domain / Alpha-2-macroglobulin, bait region domain / Alpha-macroglobulin-like, TED domain / Alpha-2-macroglobulin family / MG2 domain / A-macroglobulin TED domain / Alpha-2-macroglobulin bait region domain / Alpha-2-Macroglobulin / Alpha-2-macroglobulin family / Low-density lipoprotein receptor domain class A / Low-density lipoprotein (LDL) receptor class A, conserved site / LDL-receptor class A (LDLRA) domain signature. / LDL-receptor class A (LDLRA) domain profile. / Thrombospondin type 1 domain / Thrombospondin type-1 (TSP1) repeat superfamily / Thrombospondin type-1 (TSP1) repeat profile. / Thrombospondin type 1 repeats / Thrombospondin type-1 (TSP1) repeat / Low-density lipoprotein receptor domain class A / Low-density lipoprotein (LDL) receptor class A repeat / LDL receptor-like superfamily / Lipocalin family conserved site / Sushi repeat (SCR repeat) / Domain abundant in complement control proteins; SUSHI repeat; short complement-like repeat (SCR) / Snake toxin-like superfamily / Sushi/SCR/CCP domain / Sushi/SCR/CCP superfamily / Sushi/CCP/SCR domain profile. / Terpenoid cyclases/protein prenyltransferase alpha-alpha toroid / Lipocalin / cytosolic fatty-acid binding protein family / Lipocalin/cytosolic fatty-acid binding domain / Growth factor receptor cysteine-rich domain superfamily / EGF-like domain signature 2. / EGF-like domain signature 1. / Calycin / Lipocalin signature. / Immunoglobulin-like fold
Similarity search - Domain/homology
Complement C5 / Complement component C9 / Complement component C8 alpha chain / Complement component C8 beta chain / Complement component C8 gamma chain / Complement component C7 / Complement component C6 / CD59 glycoprotein
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsCouves, E.C. / Gardner, S. / Bubeck, D.
Funding supportEuropean Union, United Kingdom, 2items
OrganizationGrant numberCountry
European Research Council (ERC)No. 864751European Union
Cancer Research UKC24523/A26234 United Kingdom
CitationJournal: Nat Commun / Year: 2023
Title: Structural basis for membrane attack complex inhibition by CD59.
Authors: Emma C Couves / Scott Gardner / Tomas B Voisin / Jasmine K Bickel / Phillip J Stansfeld / Edward W Tate / Doryen Bubeck /
Abstract: CD59 is an abundant immuno-regulatory receptor that protects human cells from damage during complement activation. Here we show how the receptor binds complement proteins C8 and C9 at the membrane to ...CD59 is an abundant immuno-regulatory receptor that protects human cells from damage during complement activation. Here we show how the receptor binds complement proteins C8 and C9 at the membrane to prevent insertion and polymerization of membrane attack complex (MAC) pores. We present cryo-electron microscopy structures of two inhibited MAC precursors known as C5b8 and C5b9. We discover that in both complexes, CD59 binds the pore-forming β-hairpins of C8 to form an intermolecular β-sheet that prevents membrane perforation. While bound to C8, CD59 deflects the cascading C9 β-hairpins, rerouting their trajectory into the membrane. Preventing insertion of C9 restricts structural transitions of subsequent monomers and indirectly halts MAC polymerization. We combine our structural data with cellular assays and molecular dynamics simulations to explain how the membrane environment impacts the dual roles of CD59 in controlling pore formation of MAC, and as a target of bacterial virulence factors which hijack CD59 to lyse human cells.
History
DepositionSep 7, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 22, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 1, 2023Group: 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
G: CD59 glycoprotein
D: Complement component C8 beta chain
F: Complement component C8 gamma chain
E: Complement component C8 alpha chain
A: Complement C5
C: Complement component C7
B: Complement component C6
H: Complement component C9
I: Complement component C9


Theoretical massNumber of molelcules
Total (without water)682,4739
Polymers682,4739
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 2 molecules GA

#1: Protein CD59 glycoprotein / 1F5 antigen / 20 kDa homologous restriction factor / HRF-20 / HRF20 / MAC-inhibitory protein / MAC- ...1F5 antigen / 20 kDa homologous restriction factor / HRF-20 / HRF20 / MAC-inhibitory protein / MAC-IP / MEM43 antigen / Membrane attack complex inhibition factor / MACIF / Membrane inhibitor of reactive lysis / MIRL / Protectin


Mass: 14234.388 Da / Num. of mol.: 1
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 BL21 (bacteria) / References: UniProt: P13987
#5: Protein Complement C5 / Complement component 5 / C3 and PZP-like alpha-2-macroglobulin domain-containing protein 4


Mass: 188512.094 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P01031

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Complement component ... , 6 types, 7 molecules DFECBHI

#2: Protein Complement component C8 beta chain / Complement component 8 subunit beta


Mass: 67136.891 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P07358
#3: Protein Complement component C8 gamma chain


Mass: 22302.424 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P07360
#4: Protein Complement component C8 alpha chain / Complement component 8 subunit alpha


Mass: 65239.152 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P07357
#6: Protein Complement component C7


Mass: 93625.328 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P10643
#7: Protein Complement component C6


Mass: 104918.180 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P13671
#8: Protein Complement component C9


Mass: 63252.301 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P02748

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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
12C9, CD59 inhibited MAC ComplexCOMPLEXSolved in a DOPC, MSP2N2 nanodisc with a myristolated cytotopic CD59.all0MULTIPLE SOURCES
2CD59 glycoproteinCOMPLEX#11RECOMBINANT
3Complement components C5, C6, C7, C8 and C9COMPLEX#2-#81NATURAL
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDCellular location
22Homo sapiens (human)9606Serum
33Homo sapiens (human)9606
Source (recombinant)Organism: Escherichia coli BL21 (bacteria)
Buffer solutionpH: 7.4 / Details: 20 mM HEPES pH 7.4, 120 mM NaCl
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHEPES1
2120 mMsodium chlorideNaClSodium chloride1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 95 %

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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 FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 9000 nm / Nominal defocus min: 1000 nm / Calibrated defocus max: 4000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 4 / Num. of real images: 52838 / Details: Collected over 4 separate data collections

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Processing

SoftwareName: PHENIX / Version: 1.20_4459: / Classification: refinement
EM software
IDNameVersionCategory
1crYOLOparticle selection
2cryoSPARCparticle selection
3EPUimage acquisition
5CTFFIND4.1CTF correction
8ISOLDEmodel fitting
10PHENIX1.2model refinement
11cryoSPARC2initial Euler assignment
12RELIONfinal Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionDetails: Dataset 1: 737,138 Dataset 2: 1,058,026 Dataset 3: 1,330,232 Dataset 4: 722,870
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 47244 / Symmetry type: POINT
Atomic model buildingDetails: Initial fitting was done in ISODLE, final refinements were done in ISOLDE
Atomic model building
IDPDB-ID 3D fitting-ID
17NYD

7nyd

1
22J8B

2j8b

1
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00438094
ELECTRON MICROSCOPYf_angle_d0.8751537
ELECTRON MICROSCOPYf_dihedral_angle_d9.365239
ELECTRON MICROSCOPYf_chiral_restr0.0545575
ELECTRON MICROSCOPYf_plane_restr0.016740

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