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- EMDB-12646: cryoEM reconstruction of the terminal C9s and chaperone in 3C9-sMAC -

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Basic information

Entry
Database: EMDB / ID: EMD-12646
TitlecryoEM reconstruction of the terminal C9s and chaperone in 3C9-sMAC
Map dataLocally sharpened 3C9-sMAC density subtracted and focused on C9
Sample
  • Complex: 3C9-sMAC
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
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.83 Å
AuthorsMenny A / Couves EC / Bubeck D
Funding supportEuropean Union, United Kingdom, 3 items
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, 2021-
Header (metadata) releaseOct 6, 2021-
Map releaseOct 6, 2021-
UpdateNov 17, 2021-
Current statusNov 17, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.02
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12646.png

Downloads & links

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Map

FileDownload / File: emd_12646.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocally sharpened 3C9-sMAC density subtracted and focused on C9
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 300 pix.
= 314.1 Å		1.05 Å/pix.
x 300 pix.
= 314.1 Å		1.05 Å/pix.
x 300 pix.
= 314.1 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.047 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.0018206099 - 2.324569
Average (Standard dev.)0.0010185759 (±0.023277292)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 314.1 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0471.0471.047
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z314.100314.100314.100
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0022.3250.001

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Supplemental data

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Half map: Half map 1 of 3C9-sMAC density subtracted focused on C9

Fileemd_12646_half_map_1.map
AnnotationHalf map 1 of 3C9-sMAC density subtracted focused on C9
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 of 3C9-sMAC density subtracted focused on C9

Fileemd_12646_half_map_2.map
AnnotationHalf map 1 of 3C9-sMAC density subtracted focused on C9
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : 3C9-sMAC

EntireName: 3C9-sMAC
Components
  • Complex: 3C9-sMAC

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Supramolecule #1: 3C9-sMAC

SupramoleculeName: 3C9-sMAC / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#9
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
Source (natural)Organism: Homo sapiens (human)

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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

Concentration0.065 mg/mL
BufferpH: 7.4
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4-1)
Startup modelType of model: INSILICO MODEL / In silico model: Initial model generated in Relion
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.83 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 85151

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