Journal: Nat Commun / Year: 2016 Title: Structure of the poly-C9 component of the complement membrane attack complex. Authors: Natalya V Dudkina / Bradley A Spicer / Cyril F Reboul / Paul J Conroy / Natalya Lukoyanova / Hans Elmlund / Ruby H P Law / Susan M Ekkel / Stephanie C Kondos / Robert J A Goode / Georg Ramm ...Authors: Natalya V Dudkina / Bradley A Spicer / Cyril F Reboul / Paul J Conroy / Natalya Lukoyanova / Hans Elmlund / Ruby H P Law / Susan M Ekkel / Stephanie C Kondos / Robert J A Goode / Georg Ramm / James C Whisstock / Helen R Saibil / Michelle A Dunstone / Abstract: The membrane attack complex (MAC)/perforin-like protein complement component 9 (C9) is the major component of the MAC, a multi-protein complex that forms pores in the membrane of target pathogens. In ...The membrane attack complex (MAC)/perforin-like protein complement component 9 (C9) is the major component of the MAC, a multi-protein complex that forms pores in the membrane of target pathogens. In contrast to homologous proteins such as perforin and the cholesterol-dependent cytolysins (CDCs), all of which require the membrane for oligomerisation, C9 assembles directly onto the nascent MAC from solution. However, the molecular mechanism of MAC assembly remains to be understood. Here we present the 8 Å cryo-EM structure of a soluble form of the poly-C9 component of the MAC. These data reveal a 22-fold symmetrical arrangement of C9 molecules that yield an 88-strand pore-forming β-barrel. The N-terminal thrombospondin-1 (TSP1) domain forms an unexpectedly extensive part of the oligomerisation interface, thus likely facilitating solution-based assembly. These TSP1 interactions may also explain how additional C9 subunits can be recruited to the growing MAC subsequent to membrane insertion.
Mass: 57582.871 Da / Num. of mol.: 22 / Source method: isolated from a natural source / Source: (natural) HOMO SAPIENS (human) / Tissue: BLOOD / References: UniProt: P02748
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Experimental details
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Experiment
Experiment
Method: ELECTRON MICROSCOPY
EM experiment
Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction
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Sample preparation
Component
Name: POLYC9 / Type: COMPLEX
Buffer solution
Name: 10 MM TRIS, 50 MM NACL / pH: 8 / Details: 10 MM TRIS, 50 MM NACL
Specimen
Conc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen support
Details: HOLEY CARBON
Vitrification
Instrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Details: LIQUID ETHANE
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Electron microscopy imaging
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
Microscopy
Model: FEI POLARA 300 / Date: Dec 15, 2014
Electron gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lens
Mode: BRIGHT FIELD / Nominal magnification: 77000 X / Calibrated magnification: 36232 X / Nominal defocus max: 4000 nm / Nominal defocus min: 1500 nm / Cs: 2.3 mm
Specimen holder
Temperature: 85 K
Image recording
Electron dose: 25 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scans
Num. digital images: 1385
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Processing
EM software
ID
Name
Version
Category
1
CTFFIND
3
CTFcorrection
2
IMAGIC
3Dreconstruction
3
RELION
3Dreconstruction
CTF correction
Details: EACH MICROGRAPH
Symmetry
Point symmetry: C22 (22 fold cyclic)
3D reconstruction
Resolution: 6.7 Å / Num. of particles: 5000 / Nominal pixel size: 2.76 Å / Actual pixel size: 2.76 Å Details: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-3235. (DEPOSITION ID: 13993). Symmetry type: POINT
Refinement
Highest resolution: 6.7 Å
Refinement step
Cycle: LAST / Highest resolution: 6.7 Å
Protein
Nucleic acid
Ligand
Solvent
Total
Num. atoms
10582
0
0
0
10582
+
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