Structure(s) of Monomeric State of Alpha Synuclein in Solution Solved by Combining SAXS and Alphafold

SASDQJ7 – Alpha Synuclein at 283 K and concentration of 8 mg/ml

Human alpha-synuclein
MWI(0) 15 kDa
MWexpected 14 kDa
VPorod 81 nm3
log I(s) 2.20×104 2.20×103 2.20×102 2.20×101
Human alpha-synuclein small angle scattering data  s, nm-1
ln I(s)
Human alpha-synuclein Guinier plot ln 2.20×104 Rg: 4.7 nm 0 (4.7 nm)-2 s2
Human alpha-synuclein Kratky plot 1.104 0 3 sRg
Human alpha-synuclein pair distance distribution function Rg: 5.0 nm 0 Dmax: 17.6 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Human alpha-synuclein ALPHAFOLD model

log I(s)
 s, nm-1
Human alpha-synuclein ALPHAFOLD model

log I(s)
 s, nm-1
Human alpha-synuclein ALPHAFOLD model

SAXS data from solutions of alpha synuclein in 10 mM HEPES, 50 mM NaCl, pH 7.4 were were collected using an Anton Paar SAXSpace at the CSIR Institute of Microbial Technology (IMTech; Chandigarh, India) equipped with a Mythen 1K detector at a sample-detector distance of 0.3 m and at a wavelength of λ = 0.15414 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). One solute concentration of 8.00 mg/ml was measured at 10°C. Three successive 1800 second frames were collected. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

CAUTION: Experimental errors are incorrectly specified (negative values). CAUTION: Experimental molecular weight cannot be verified from the data. The human wild-type α-syn protein was expressed in E. coli Rosetta 2 (DE3) using pT7-7 based expression vector. The protein expression was induced using 0.3mM IPTG for 8h at 30 °C. Cells were harvested by centrifugation and further resuspended into buffer containing in10 mM HEPES, 50 mM NaCl, (pH 7.4). The harvested cell pellet was stored at -80 °C until further use. Briefly, cell lysis was carried out by sonication, and cellular lysate was boiled at 95 °C for 30 minutes. Streptomycin sulfate and Glacial acetic acid precipitated nucleic acid material was removed by centrifugation at 12000 r.p.m. for 30 minutes at 4 °C. The α-syn was precipitated by addition of 50% ammonium sulphate followed by incubation on ice for 1 h with regular shaking after every 10 minutes. The pellet was separated, and further washed with equal volume of 100 mM ammonium acetate followed by equal volume of ethanol. The washed protein pellet was dried to evaporate any residual ethanol and further dissolved in10 mM HEPES, 50 mM NaCl, pH 7.4 and extensively dialyzed to remove ammonium sulphate. Protein purity was confirmed on 15% SDS-PAGE.

Human alpha-synuclein (aSyn)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   14.5 kDa
Sequence   FASTA