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- PDB-7nyc: cryoEM structure of 3C9-sMAC -

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Basic information

Entry
Database: PDB / ID: 7nyc
TitlecryoEM structure of 3C9-sMAC
Components
  • (Complement component ...) x 6
  • Complement C5Complement component 5
KeywordsIMMUNE SYSTEM / Complement / MACPF / Membrane Attack Complex / CDC / pore forming
Function / homology
Function and homology information


cell killing / Terminal pathway of complement / membrane attack complex / complement binding / other organism cell membrane / Activation of C3 and C5 / negative regulation of macrophage chemotaxis / complement activation, alternative pathway / complement activation / chemokine activity ...cell killing / Terminal pathway of complement / membrane attack complex / complement binding / other organism cell membrane / Activation of C3 and C5 / negative regulation of macrophage chemotaxis / complement activation, alternative pathway / complement activation / chemokine activity / retinol binding / endopeptidase inhibitor activity / positive regulation of vascular endothelial growth factor production / complement activation, classical pathway / positive regulation of chemokine production / Peptide ligand-binding receptors / Regulation of Complement cascade / protein homooligomerization / extracellular vesicle / chemotaxis / positive regulation of immune response / G alpha (i) signalling events / blood microparticle / killing of cells of another organism / in utero embryonic development / cell surface receptor signaling pathway / immune response / inflammatory response / G protein-coupled receptor signaling pathway / signaling receptor binding / innate immune response / protein-containing complex binding / 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 / Membrane attack complex component/perforin/complement C9 / 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. / : / 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) / 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
beta-D-mannopyranose / alpha-L-fucopyranose / 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
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.54 Å
AuthorsMenny, A. / Couves, E.C. / Bubeck, D.
Funding supportEuropean Union, United Kingdom, 3items
OrganizationGrant numberCountry
European Research Council (ERC)No. 864751European Union
Cancer Research UKC24523/A26234 United Kingdom
Engineering and Physical Sciences Research CouncilEP/L015498/1 United Kingdom
CitationJournal: Nat Commun / Year: 2021
Title: Structural basis of soluble membrane attack complex packaging for clearance.
Authors: Anaïs Menny / Marie V Lukassen / Emma C Couves / Vojtech Franc / Albert J R Heck / Doryen Bubeck /
Abstract: Unregulated complement activation causes inflammatory and immunological pathologies with consequences for human disease. To prevent bystander damage during an immune response, extracellular ...Unregulated complement activation causes inflammatory and immunological pathologies with consequences for human disease. To prevent bystander damage during an immune response, extracellular chaperones (clusterin and vitronectin) capture and clear soluble precursors to the membrane attack complex (sMAC). However, how these chaperones block further polymerization of MAC and prevent the complex from binding target membranes remains unclear. Here, we address that question by combining cryo electron microscopy (cryoEM) and cross-linking mass spectrometry (XL-MS) to solve the structure of sMAC. Together our data reveal how clusterin recognizes and inhibits polymerizing complement proteins by binding a negatively charged surface of sMAC. Furthermore, we show that the pore-forming C9 protein is trapped in an intermediate conformation whereby only one of its two transmembrane β-hairpins has unfurled. This structure provides molecular details for immune pore formation and helps explain a complement control mechanism that has potential implications for how cell clearance pathways mediate immune homeostasis.
History
DepositionMar 22, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 6, 2021Provider: repository / Type: Initial release
Revision 1.1Nov 17, 2021Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_database_proc
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

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  • Deposited structure unit
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  • EMDB-12650
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Structure viewerMolecule:
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Assembly

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


Theoretical massNumber of molelcules
Total (without water)710,23626
Polymers706,7959
Non-polymers3,44117
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area61330 Å2
ΔGint-69 kcal/mol
Surface area241430 Å2
MethodPISA

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Components

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Complement component ... , 6 types, 8 molecules CDEGHIBF

#1: Protein Complement component C7


Mass: 91221.484 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P10643
#2: Protein Complement component C8 beta chain / Complement component 8 subunit beta


Mass: 61122.852 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 alpha chain / Complement component 8 subunit alpha


Mass: 61782.992 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P07357
#4: Protein Complement component C9


Mass: 61056.594 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P02748
#5: Protein Complement component C6


Mass: 102541.312 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P13671
#7: Protein Complement component C8 gamma chain


Mass: 20410.105 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P07360

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Protein / Non-polymers , 2 types, 4 molecules A

#11: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Ca
#6: Protein Complement C5 / Complement component 5 / C3 and PZP-like alpha-2-macroglobulin domain-containing protein 4


Mass: 186546.656 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P01031

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Sugars , 5 types, 14 molecules

#8: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#9: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}}LINUCSPDB-CARE
#10: Sugar
ChemComp-BMA / beta-D-mannopyranose / beta-D-mannose / D-mannose / mannose / Mannose


Type: D-saccharide, beta linking / Mass: 180.156 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C6H12O6
IdentifierTypeProgram
DManpbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
b-D-mannopyranoseCOMMON NAMEGMML 1.0
b-D-ManpIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
ManSNFG CARBOHYDRATE SYMBOLGMML 1.0
#12: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#13: Sugar ChemComp-FUC / alpha-L-fucopyranose / alpha-L-fucose / 6-deoxy-alpha-L-galactopyranose / L-fucose / fucose / Fucose


Type: L-saccharide, alpha linking / Mass: 164.156 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H12O5
IdentifierTypeProgram
LFucpaCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
a-L-fucopyranoseCOMMON NAMEGMML 1.0
a-L-FucpIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
FucSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Details

Has ligand of interestN

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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: 3C9-sMAC / Type: COMPLEX
Details: This sMAC oligomer contains one copy of C5b8 and 3 copies of C9, as well as multiple copies of the chaperones vitronectin and clusterin
Entity ID: #1-#7 / Source: NATURAL
Molecular weightValue: 1 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
SpecimenConc.: 0.065 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 95 % / 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 FIELDBright-field microscopy / C2 aperture diameter: 70 µm
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0253 / Classification: refinement
EM software
IDNameVersionCategory
2crYOLOparticle selection
3EPUimage acquisition
5CTFFIND4-1CTF correction
8UCSF ChimeraXmodel fitting
9Coot9.1model fitting
11RELION3.1initial Euler assignment
12RELION3.1final Euler assignment
14RELION3.13D reconstruction
15ISOLDEmodel refinement
16Coot9.1model refinement
17REFMACmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.54 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 85151 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model building
IDPDB-ID 3D fitting-ID
16H03

6h03

1
26H04

6h04

1
34A5W

4a5w

1
42WCY

2wcy

1
56CXO

6cxo

1
RefinementResolution: 3.5→418.8 Å / Cor.coef. Fo:Fc: 0.891 / SU B: 16.753 / SU ML: 0.236 / ESU R: 0.166
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
RfactorNum. reflection% reflection
Rwork0.4316 --
obs0.4316 3588767 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 268.658 Å2
Baniso -1Baniso -2Baniso -3
1-4.12 Å2-7.2 Å2-11.7 Å2
2---4.69 Å24.56 Å2
3---0.57 Å2
Refinement stepCycle: 1 / Total: 41391
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0130.01242358
ELECTRON MICROSCOPYr_bond_other_d
ELECTRON MICROSCOPYr_angle_refined_deg2.0961.64957454
ELECTRON MICROSCOPYr_angle_other_deg
ELECTRON MICROSCOPYr_dihedral_angle_1_deg10.33755284
ELECTRON MICROSCOPYr_dihedral_angle_2_deg32.64822.652211
ELECTRON MICROSCOPYr_dihedral_angle_3_deg12.117156945
ELECTRON MICROSCOPYr_dihedral_angle_4_deg17.98515258
ELECTRON MICROSCOPYr_chiral_restr0.1330.25570
ELECTRON MICROSCOPYr_gen_planes_refined0.0170.0232539
ELECTRON MICROSCOPYr_gen_planes_other
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it87.15824.75721220
ELECTRON MICROSCOPYr_mcbond_other
ELECTRON MICROSCOPYr_mcangle_it36.53226476
ELECTRON MICROSCOPYr_mcangle_other
ELECTRON MICROSCOPYr_scbond_it28.47121138
ELECTRON MICROSCOPYr_scbond_other
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other
ELECTRON MICROSCOPYr_long_range_B_refined161521
ELECTRON MICROSCOPYr_long_range_B_other
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.5→3.591 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.798 265610 -
obs--100 %

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