Decoupling of size and shape fluctuations in heteropolymeric sequences reconciles discrepancies in SAXS vs. FRET measurements.

Fuertes G, Banterle N, Ruff KM, Chowdhury A, Mercadante D, Koehler C, Kachala M, Estrada Girona G, Milles S, Mishra A, Onck PR, Gräter F, Esteban-Martín S, Pappu RV, Svergun DI, Lemke EA, Proc Natl Acad Sci U S A 114(31):E6342-E6351 (2017) Europe PMC

SASDEU2 – Labeled Importin Beta Binding Domain from importin subunit alpha-1 (IBB-Alexa488/Alexa594) with denaturant

Importin subunit alpha-1
Alexa Fluor™ 594 C5 Maleimide
Alexa Fluor™ 488 C5 Hydroxylamine
MWexperimental 11 kDa
MWexpected 13 kDa
VPorod 29 nm3
log I(s) 4.19×102 4.19×101 4.19×100 4.19×10-1
Importin subunit alpha-1 Alexa Fluor™ 594 C5 Maleimide Alexa Fluor™ 488 C5 Hydroxylamine small angle scattering data  s, nm-1
ln I(s)
Importin subunit alpha-1 Alexa Fluor™ 594 C5 Maleimide Alexa Fluor™ 488 C5 Hydroxylamine Guinier plot ln 4.20×102 Rg: 3.2 nm 0 (3.2 nm)-2 s2
(sRg)2I(s)/I(0)
Importin subunit alpha-1 Alexa Fluor™ 594 C5 Maleimide Alexa Fluor™ 488 C5 Hydroxylamine Kratky plot 1.104 0 3 sRg
p(r)
Importin subunit alpha-1 Alexa Fluor™ 594 C5 Maleimide Alexa Fluor™ 488 C5 Hydroxylamine pair distance distribution function Rg: 3.4 nm 0 Dmax: 13.3 nm

Data validation


There are no models related to this curve.

Synchrotron SAXS data from solutions of Labeled Importin Beta Binding Domain from importin subunit alpha-1 (IBB-Alexa488/Alexa594) with denaturant in PBS, 10 mM DTT, 6 M urea, 0.3 M KCl, pH 7.4 were collected on the EMBL P12 beam line at the PETRA III storage ring (Hamburg, Germany) using a Pilatus 2M detector at a sample-detector distance of 3 m and at a wavelength of λ = 0.1 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). One solute concentration of 10.00 mg/ml was measured at 23°C. 20 successive 0.050 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.

The protein contains a penultimate non-canonical amino acid p-acetylphenylalanine (207 Da) that is represented as U (selenocysteine, 168 Da) in the amino acid sequence for the entry. Therefore, the calculated MW from sequence (MW(expected)) must be adjusted accordingly (ca. 40 Da).

Tags: idp
Importin subunit alpha-1 (IBB-488/594)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   11.3 kDa
 
UniProt   P52292 (3-97)
Sequence   FASTA
 
Alexa Fluor™ 594 C5 Maleimide (Alexa594)
Mol. type   Other
Olig. state   Monomer
Mon. MW   0.9 kDa
Alexa Fluor™ 488 C5 Hydroxylamine (Alexa488)
Mol. type   Other
Olig. state   Monomer
Mon. MW   0.7 kDa