The dynamic nature of netrin-1 and the structural basis for glycosaminoglycan fragment-induced filament formation

Meier M, Gupta M, Akgül S, McDougall M, Imhof T, Nikodemus D, Reuten R, Moya-Torres A, To V, Ferens F, Heide F, Padilla-Meier G, Kukura P, Huang W, Gerisch B, Mörgelin M, Poole K, Antebi A, Koch M, Stetefeld J, Nature Communications 14(1) (2023) DOI

SASDRQ2 – Chicken Netrin-1 ΔC bound to heparin oligosaccharide dp10 (experiment ID: sm16028-7/379533)

Heparin oligosaccharide dp10 ammonium salt
MWexperimental 333 kDa
MWexpected 251 kDa
VPorod 758 nm3
log I(s) 2.34×10-1 2.34×10-2 2.34×10-3 2.34×10-4
Netrin-1 Heparin oligosaccharide dp10 ammonium salt small angle scattering data  s, nm-1
ln I(s)
Netrin-1 Heparin oligosaccharide dp10 ammonium salt Guinier plot ln 2.34×10-1 Rg: 6.4 nm 0 (6.4 nm)-2 s2
Netrin-1 Heparin oligosaccharide dp10 ammonium salt Kratky plot 1.104 0 3 sRg
Netrin-1 Heparin oligosaccharide dp10 ammonium salt pair distance distribution function Rg: 6.6 nm 0 Dmax: 21.7 nm

Data validation

Fits and models

log I(s)
 s, nm-1

log I(s)
 s, nm-1

log I(s)
 s, nm-1

SEC-SAXS data were collected at beamline B21, Diamond Light Source (Didcot, UK) that operates at a wavelength of 0.1 nm at a photon flux of ~10¹² photons per second and features a fixed camera length of 4.014 m. It is equipped with an Agilent 1200 HPLC system. Tag-free chicken Netrin-1 ΔC was shipped to the beamline at 1.0 mg/ml in 50 mM tris, pH 7.5, 1.0 M NaCl. Using a PD-10 desalting column with Sephadex G-25 resin, the buffer was changed to 50 mM tris, pH 7.5, 0.2 M NaCl and the protein was then concentrated to 9.23 ± 0.09 mg/ml (186 ± 2 µM) using an Amicon-4 concentrator with 30 kDa molecular weight cut-off. 20 µl of heparin oligosaccharide dp10 (10 mg/ml in water) was added to 100 µl protein solution and let to incubate for 40 - 80 min before injection.The sample was then centrifuged at 13000 rpm in an Eppendorf MiniSpin® centrifuge followed by filtration through a 0.1 µM Amicon Ultrafree-MC spin filter. 50 µl of sample were injected without further delay into the buffer equilibrated 4.6 ml Shodex KW 403-4F size exclusion column from where the protein eluted through the diffraction flow cell at a rate of 0.16 ml/min. X-ray images were collected with 3 s exposure time.

Experiment ID: sm16028-7/379533. Additional modelling information, including log files, are provided in the full entry zip archive. Each respective fit displays two maps: 1) The refined averaged map (top) and; 2) The corresponding object support (bottom).

Netrin-1 (NET1 ΔC)
Mol. type   Protein
Organism   Gallus gallus
Olig. state   Pentamer
Mon. MW   49.5 kDa
UniProt   Q90922 (26-458)
Sequence   FASTA
Heparin oligosaccharide dp10 ammonium salt (dp10)
Mol. type   Other
Organism   Sus scrofa domesticus
Olig. state   Monomer
Mon. MW   3 kDa
Chemical formula