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

SASDEL2 – Labeled nucleoporin NUP49/NSP49 (N49-Alexa488/Alexa594) with denaturant

Nucleoporin NUP49/NSP49
Alexa Fluor™ 594 C5 Maleimide
Alexa Fluor™ 488 C5 Hydroxylamine
MWexperimental 6 kDa
MWexpected 5 kDa
VPorod 7 nm3
log I(s) 6.37×102 6.37×101 6.37×100 6.37×10-1
Nucleoporin NUP49/NSP49 Alexa Fluor™ 594 C5 Maleimide Alexa Fluor™ 488 C5 Hydroxylamine small angle scattering data  s, nm-1
ln I(s)
Nucleoporin NUP49/NSP49 Alexa Fluor™ 594 C5 Maleimide Alexa Fluor™ 488 C5 Hydroxylamine Guinier plot ln 6.38×102 Rg: 2.1 nm 0 (2.1 nm)-2 s2
(sRg)2I(s)/I(0)
Nucleoporin NUP49/NSP49 Alexa Fluor™ 594 C5 Maleimide Alexa Fluor™ 488 C5 Hydroxylamine Kratky plot 1.104 0 3 sRg
p(r)
Nucleoporin NUP49/NSP49 Alexa Fluor™ 594 C5 Maleimide Alexa Fluor™ 488 C5 Hydroxylamine pair distance distribution function Rg: 2.2 nm 0 Dmax: 7.7 nm

Data validation


There are no models related to this curve.

Synchrotron SAXS data from solutions of Labeled nucleoporin NUP49/NSP49 (N49-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 (DESY; 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 8.50 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
Nucleoporin NUP49/NSP49 (N49-488/594)
Mol. type   Protein
Organism   Saccharomyces cerevisiae
Olig. state   Monomer
Mon. MW   3.9 kDa
 
UniProt   Q02199 (121-154)
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