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- EMDB-7773: The X-ray crystal structure of Complement component-9 reveals tha... -

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Basic information

Entry
Database: EMDB / ID: EMD-7773
TitleThe X-ray crystal structure of Complement component-9 reveals that the first trans-membrane region acts as a brake on self-assembly
Map dataStructure of the polyC9 component of the membrane attack complex.
Sample
  • Organelle or cellular component: polyC9
    • Protein or peptide: human complement protein 9
Function / homology
Function and homology information


cell killing / Terminal pathway of complement / membrane attack complex / other organism cell membrane / complement activation, alternative pathway / complement activation / complement activation, classical pathway / Regulation of Complement cascade / protein homooligomerization / positive regulation of immune response ...cell killing / Terminal pathway of complement / membrane attack complex / other organism cell membrane / complement activation, alternative pathway / complement activation / complement activation, classical pathway / Regulation of Complement cascade / protein homooligomerization / positive regulation of immune response / blood microparticle / killing of cells of another organism / extracellular space / extracellular exosome / extracellular region / plasma membrane
Similarity search - Function
Membrane attack complex component/perforin/complement C9 / Membrane attack complex component/perforin domain, conserved site / Membrane attack complex/perforin (MACPF) domain signature. / membrane-attack complex / perforin / Membrane attack complex/perforin (MACPF) domain profile. / MAC/Perforin domain / Membrane attack complex component/perforin (MACPF) domain / Low-density lipoprotein receptor domain class A / Low-density lipoprotein (LDL) receptor class A, conserved site / LDL-receptor class A (LDLRA) domain signature. ...Membrane attack complex component/perforin/complement C9 / Membrane attack complex component/perforin domain, conserved site / Membrane attack complex/perforin (MACPF) domain signature. / membrane-attack complex / perforin / Membrane attack complex/perforin (MACPF) domain profile. / MAC/Perforin domain / Membrane attack complex component/perforin (MACPF) domain / 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 / Growth factor receptor cysteine-rich domain superfamily / EGF-like domain signature 2. / EGF-like domain signature 1.
Similarity search - Domain/homology
Complement component C9
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsSpicer BA / Law RHP / Pang S / Dunstone MA / Whisstock JC
CitationJournal: Nat Commun / Year: 2018
Title: The first transmembrane region of complement component-9 acts as a brake on its self-assembly.
Authors: Bradley A Spicer / Ruby H P Law / Tom T Caradoc-Davies / Sue M Ekkel / Charles Bayly-Jones / Siew-Siew Pang / Paul J Conroy / Georg Ramm / Mazdak Radjainia / Hariprasad Venugopal / James C ...Authors: Bradley A Spicer / Ruby H P Law / Tom T Caradoc-Davies / Sue M Ekkel / Charles Bayly-Jones / Siew-Siew Pang / Paul J Conroy / Georg Ramm / Mazdak Radjainia / Hariprasad Venugopal / James C Whisstock / Michelle A Dunstone /
Abstract: Complement component 9 (C9) functions as the pore-forming component of the Membrane Attack Complex (MAC). During MAC assembly, multiple copies of C9 are sequentially recruited to membrane associated ...Complement component 9 (C9) functions as the pore-forming component of the Membrane Attack Complex (MAC). During MAC assembly, multiple copies of C9 are sequentially recruited to membrane associated C5b8 to form a pore. Here we determined the 2.2 Å crystal structure of monomeric murine C9 and the 3.9 Å resolution cryo EM structure of C9 in a polymeric assembly. Comparison with other MAC proteins reveals that the first transmembrane region (TMH1) in monomeric C9 is uniquely positioned and functions to inhibit its self-assembly in the absence of C5b8. We further show that following C9 recruitment to C5b8, a conformational change in TMH1 permits unidirectional and sequential binding of additional C9 monomers to the growing MAC. This mechanism of pore formation contrasts with related proteins, such as perforin and the cholesterol dependent cytolysins, where it is believed that pre-pore assembly occurs prior to the simultaneous release of the transmembrane regions.
History
DepositionApr 2, 2018-
Header (metadata) releaseMay 9, 2018-
Map releaseSep 19, 2018-
UpdateOct 3, 2018-
Current statusOct 3, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6dlw
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6dlw
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7773.png

Downloads & links

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Map

FileDownload / File: emd_7773.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of the polyC9 component of the membrane attack complex.
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.035 / Movie #1: 0.035
Minimum - Maximum-0.1444413 - 0.21477337
Average (Standard dev.)0.00077728054 (±0.0068512256)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions350350350
Spacing350350350
CellA=B=C: 370.99997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z350350350
origin x/y/z0.0000.0000.000
length x/y/z371.000371.000371.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS350350350
D min/max/mean-0.1440.2150.001

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Supplemental data

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Mask #1

Fileemd_7773_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Complement component-9, Combined unfiltered raw map.

Fileemd_7773_additional.map
AnnotationComplement component-9, Combined unfiltered raw map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Complement component-9, Unfiltered half map 2.

Fileemd_7773_half_map_1.map
AnnotationComplement component-9, Unfiltered half map 2.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Complement component-9, Unfiltered half map 1.

Fileemd_7773_half_map_2.map
AnnotationComplement component-9, Unfiltered half map 1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : polyC9

EntireName: polyC9
Components
  • Organelle or cellular component: polyC9
    • Protein or peptide: human complement protein 9

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Supramolecule #1: polyC9

SupramoleculeName: polyC9 / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all / Details: polyC9
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 1342 kDa/nm
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: Expi293 / Recombinant plasmid: pSEctag2a

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Macromolecule #1: human complement protein 9

MacromoleculeName: human complement protein 9 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: msacrsfav a icileisi lt aqyttsy dpe ltessg sash idcrm spwse wsqc dpclrq mfr srsievf gq fngkrctd a vgdrrqcvp tepcedaedd cgndfqcst g rcikmrlr cn gdndcgd fsd eddces eprp pcrdr vvees elar ...String:
msacrsfav a icileisi lt aqyttsy dpe ltessg sash idcrm spwse wsqc dpclrq mfr srsievf gq fngkrctd a vgdrrqcvp tepcedaedd cgndfqcst g rcikmrlr cn gdndcgd fsd eddces eprp pcrdr vvees elar tagygi nil gmdplst pf dnefyngl c nrdrdgntl tyyrrpwnva sliyetkge k nfrtehye eq ieafksi iqe ktsnfn aais lkftp tetnk aeqc ceetas sis lhgkgsf rf sysknety q lflsysskk ekmflhvkge ihlgrfvmr n rdvvlttt fv ddikalp tty ekgeyf afle tygth ysssg slgg lyeliy vld kasmkrk gv elkdikrc l gyhldvsla fseisvgaef nkddcvkrg e gravnits en liddvvs lir ggtrky afel kekll rgtvi dvtd fvnwas sin dapvlis qk lspiynlv p vkmknahlk kqnleraied yinefsvrk c htcqnggt vi lmdgkcl cac pfkfeg iace iskqk isegl pale fpnek

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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

Concentration1.3 mg/mL
BufferpH: 7.2
Component:
ConcentrationName
10.0 mMHEPES
50.0 mMsodium chloride
0.02 mg/mlamohipol A8-35
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details: 2.5 uL sample applied to Quantifoil grid R1.2/1.3 x200 mesh.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Number grids imaged: 1 / Average exposure time: 8.0 sec. / Average electron dose: 46.4 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 220000
Startup modelType of model: OTHER / Details: Ab initio model
Initial angle assignmentType: COMMON LINE
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. v2.1b.1)
Final reconstructionApplied symmetry - Point group: C22 (22 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. v2.1b.1) / Number images used: 58000
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: BACKBONE TRACE
Output model

PDB-6dlw:
Complement component polyC9

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