The allosteric modulation of Complement C5 by knob domain peptides.

Macpherson A, Laabei M, Ahdash Z, Graewert MA, Birtley JR, Schulze ME, Crennell S, Robinson SA, Holmes B, Oleinikovas V, Nilsson PH, Snowden J, Ellis V, Mollnes TE, Deane CM, Svergun D, Lawson AD, van den Elsen JM, Elife 10 (2021) Europe PMC

SASDJA6 – Complement C5

Complement C5
MWexperimental 188 kDa
MWexpected 186 kDa
VPorod 392 nm3
log I(s) 5.31×104 5.31×103 5.31×102 5.31×101
Complement C5 small angle scattering data  s, nm-1
ln I(s)
Complement C5 Guinier plot ln 5.32×104 Rg: 4.8 nm 0 (4.8 nm)-2 s2
Complement C5 Kratky plot 1.104 0 3 sRg
Complement C5 pair distance distribution function Rg: 4.9 nm 0 Dmax: 17.6 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Complement C5 CUSTOM IN-HOUSE model

log I(s)
 s, nm-1
Complement C5 SREFLEX model

Data was collected at the EMBL P12 beam line (PETRA III, DESY Hamburg, Germany). Data was collected with inline in size exclusion chromatography (SEC) mode using the Agilent 1260 Infinity II Bio-inert LC system at the beamline. 50 μL of complement component C5 at 31.6 μM (5.96 mg/ml) were injected onto a Superdex 200 Increase 5/150 column (GE Healthcare) at a flow rate of 0.35 ml/min. The mobile phase was comprised of 20mM Tris pH 7.35, 75mM NaCl, and 3% glycerol. The column elute was directly streamed to the SAXS capillary cell, and throughout the 15-minute-run 900 frames of 1 sec exposure were collected. After data reduction and radial averaging the program CHROMIXS was employed. Around 100 statistically similar buffer frames were selected and used for background subtraction of the sample frames from the chromatographic peak. This results in the final I(s) vs s scattering profiles, where s = 4πsinθ/λ, 2θ is the scattering angle and λ = 1.24 Å. The scattering data in the momentum transfer range 0.05 < s < 0.32 nm-1 were collected with a PILATUS 6M pixel detector at a distance of 3.1 m from the sample. On the same day, multi-angle laser light scattering (MALLS) data was collected with a separate SEC run yet under the same experimental conditions (set-up, buffer, run parameters etc.).A Wyatt Technologies miniDAWN TREOS multiangle laser light scattering detector was used, coupled to an OptiLab T-Rex differential refractometer for protein concentration determination (dn/dc was taken as 0.185). The MALLS system was calibrated relative to the scattering from toluene. In addition to the apo entry, ligated versions as described in the manuscript are available as additional files.

Complement C5 (C5)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   186.3 kDa
UniProt   P01031 (19-1676)
Sequence   FASTA
PDB code   5HCC
PDB code   5HCC